Prevention and modulation of aminoglycoside ototoxicity (Review)

More than 60 years after their isolation and characterization, aminoglycoside (AG) antibiotics remain powerful agents in the treatment of severe gram-negative, enterococcal or mycobacterial infections. However, the clinical use of AGs is hampered by nephrotoxicity and ototoxicity, which often develop as a consequence of prolonged courses of therapy, or of administration of increased doses of these drugs. The discovery of non-ototoxic antibacterial agents, showing a wider spectrum of activity, has gradually decreased the use of AGs as first line antibiotics for many systemic infections. However, AGs are now undergoing an unexpected revival, being increasingly indicated for the treatment of severe emerging infections caused by organisms showing resistance to most first-line agents (e.g., multidrug-resistant tuberculosis, complicated nosocomially-acquired acute urinary tract infections). Increasing adoption of aminoglycosides poses again to scientists and physicians the problem of toxicity directed to the kidneys and to the inner ear. In particular, aminoglycoside-induced deafness can be profound and irreversible, especially in genetically predisposed patients. For this reason, an impressive amount of molecular strategies have been developed in the last decade to counteract the ototoxic effect of aminoglycosides. The present article overviews: i) the molecular mechanisms by which aminoglycosides exert their bactericidal activity, ii) the mechanisms whereby AGs exert their ototoxic activity in genetically-predisposed patients, iii) the drugs and compounds that have so far proven to prevent or modulate AG ototoxicity at the preclinical and/or clinical level, and iv) the dosage regimens that have so far been suggested to decrease the incidence of episodes of AG-induced ototoxicity

Molecular analysis of PDGFRA and PDGFRB genes by rapid single-strand conformation polymorfism (SSCP) in patients with core-binding factor leukaemias with KIT or FLT3 mutation

BACKGROUND: Mutations involving KIT and FLT3 genes, encoding tyrosine kinase (TK) membrane receptors, are detected in core-binding factor leukaemia (CBFL) patients. PDFGRA and PDGFRB encode class III TK receptors and are involved both in physiological processes and in the pathogenesis of haematological and solid tumours. The aim of this study was to investigate if PDGFR mutations are involved in CBFL. PATIENTS AND METHODS: In order to detect PDGFR mutations in CBFL, 35 patients without KIT or FLT3 mutations patients were screened by rapid and sensitive single-strand conformation polymorphism (SSCP) analysis. Sequence analysis was performed in polymerase chain reaction (PCR) products showing altered mobility in SSCP analysis in order to determine the nucleotide changes. RESULTS: Three types of single-nucleotide polymorphism (SNP) were detected in the PDGFRA gene (exon 12, exon 13 and exon 18) while no mutation of PDGFRB was detected in the tested CBFLs. CONCLUSION: These data showed that no pathogenic mutations in PDGFRA and PDGFRB were detected in the context of CBFL without KIT and FLT3 mutations. Thus, PDGFR genes do not seem to be involved in CBFL and future studies are needed to establish the genetic causes of the disease in these particular patients

Frequency of butyrylcholinesterase gene mutations in individuals with abnormal inhibition numbers: an Italian population study

Objectives: More than 30 genetic variants of serum cholinesterase (butyrylcholinesterase, BChE) have been described. Some of them (the atypical and the fluoride-resistant variants) are well known because carriers are prone to develop prolonged apnea following the administration of the muscle relaxant succinylcholine. Genotype characterization is therefore important in order to prevent such episodes. Genetic studies have so far focused on selected individuals or families rather than on the random population. Methods: From a large group of healthy blood donors (n = 2609), we selected all the 58 individuals with low serum cholinesterase activity: among them 28 subjects had abnormal dibucaine and fluoride inhibition numbers. Twenty-five mutations in the coding region of the human cholinesterase gene were analyzed. Results: All individuals with abnormal inhibition numbers were homozygotes or double heterozygotes in several mutafions. Asp70Gly (Atypical variant) and Ala539Thr (K variant) were the most frequently observed amino acid substitutions. The majority of subjects with low BChE activity but normal dibucaine and fluoride number presented only the K form. We analyzed 106 randomly chosen subjects for K and atypical variants. Carriers of these alleles were at risk of Iow BChE activity (OR = 9.55, 95%Cl, 5.61-16.26 and OR = 30.33, 95%CI, 7.05-130.52 respectively). Conclusions: Data obtained from this study help to better define the etiology of low BChE activity and the role of the rather common K allele. It is the first time that such a large population has been screened for so many mutations. BChE is also implicated in detoxifying cocaine; therefore genetic analysis could be useful in cases of cocaine toxicity in Italian subjects

Assay using succinyldithiocholine as substrate: the method of choice for the measurement of cholinesterase catalytic activity in serum to diagnose succinyldicholine sensitivity

No comparative information is available concerning the ability of various cholinesterase (ChE) methods to identify succinyldicholine-sensitive patients, purely on the basis of the enzyme activity recorded in serum. Here, we evaluated six different methods for the measurement of ChE activity; 131 subjects were subdivided according to ChE phenotype and, therefore, to succinyldicholine sensitivity. ChE phenotype was determined by measuring dibucaine and fluoride numbers. DNA analysis was also performed to confirm correlation between the phenotype classification used in the study and the ChE genotype. The tested methods were significantly different in their ability to discriminate between the subjects with and without succinyldicholine-sensitive phenotypes. The succinyldithiocholine/5,5\u2032-dithio-bis(2-nitrobenzoate) (DTNB) method showed the highest accuracy (area under the receiver operating characteristic (ROC) curve 0.97) followed by the propionylthiocholine/DTNB method (area under the ROC curve 0.94). On the other hand, the two methods using butyrylthiocholine as substrate and that employing benzoylcholine showed limited clinical utility in discriminating subjects at risk of prolonged apnea (area under the ROC curve 640.9). Using the succinyldithiocholine method, a value 6423 U/l was approximately five times as likely to occur in a sensitive individual as in a normal one

Genetic variations within KRIT1/CCM1, MGC4607/CCM2 and PDCD10/CCM3 in a large italian family harbouring a krit1/CCM1 mutation

Cerebral cavernous malformations (CCMs) are congenital vascular anomalies of the central nervous system that can result in seizures, haemorrhage, recurrent headaches and focal neurologic deficit. CCMs can occur as an autosomal dominant trait with incomplete penetrance and a wide phenotypic variability. The genes responsible for this disease are KRIT1/CCM1 on chromosome 7q21.2, MGC4607/CCM2 on chromosome 7p15-p13 and PDCD10/CCM3 on chromosome 3q25.2-q27. Mutations in KRIT1/CCM1 account for more than 40% of CCMs. We previously reported a CCM family harbouring the KRIT1/CCM1 1204delAACAA mutation. In order to search for possible explanation of the clinical variability observed, we looked for genetic variation within exons and exon/intron regions in the three genes KRIT1, MGC4607 and PDCD10 associated to the disease within this large family, 23 subjects have been analysed. Identified genetic variations in the three genes are here presented. We believe that genetic variations could interfere with the proper CCM1/CCM2/CCM3 protein complex thus explaining the observed clinical variability

Familial cerebral cavernous malformation : report of a further Italian family

Cerebral cavernous malformations (CCMs) are vascular abnormalities that may cause seizures, headaches, intracerebral hemorrhages, and focal neurological deficits; they can also be clinically silent and may occur as a sporadic or an autosomal dominant condition. Three genes have been identified as causing familial CCM: KRIT1/CCM1, MGC4607/CCM2, and PDCD10/ CCM3, mapping, respectively, on chromosomes 7q, 7p, and 3q. This is a report on an Italian family affected by CCM due to a KRIT1 gene mutation on exon 13. The mother suffered from a cerebellar hematoma and was severely disabled; one son had suffered from intractable seizures and underwent surgery for removal of a cavernous angioma, while another son was asymptomatic. Brain MRI showed CCMs in all patients. This report underlines that a familial form of CCM could be suspected when a patient presents with multiple CCMs; neurologists and neurosurgeons should be aware that genetic testing for these forms is available

Phenotypic heterogeneity in a SOD1 G93D Italian ALS family : an example of human model to study a complex disease

We report different clinical expression in seven members of a large family with amyotrophic lateral sclerosis (ALS) and the G93D mutation in exon 4 of the Cu/Zn superoxide dismutase (SOD1) gene. The ALS clinical course in the proband showed an unusually fast progression of the disease compared to the paucisymptomatic presentation associated to this mutation in the two previously Italian families described. The remaining mutation carriers did not show the aggressive clinical course displayed by the proband. We selected few genes known to be ALS modifiers searching for genetic variants that could explain the wide phenotypic diversity within the family. Exclusion of causative genes such as TDP43, FUS, PGRN and VAPB was performed too. We believe that this kind of family with contrasting phenotypes of ALS may be considered an excellent human model to study the relationship between a wider genetic profile, including modifier genes, and the clinical expression of the disease. Therefore, the novelty of our approach is also represented by the study of a single family to reproduce a composite structure in which search for possible modifier genes/genetic variants linked to SOD1 mutated

Effect of incremental doses of folate on homocysteine and metabolically related vitamin concentrations in nondiabetic patients on peritoneal dialysis

The role of folate supplementation in reducing hyperhomocystinemia in patients on dialysis has been reported, but the optimal dose of folate is still unknown. The aim of the present study was to investigate whether greater than 5 mg/day folate supplementation provides any additional effect on plasma homocysteine (HCY) levels. The study was prospective, open, and had no control group. Of the 64 eligible nondiabetic patients on peritoneal dialysis with hyperhomocystinemia (>20 mol/L), 56 were given oral folate (5 mg/day) for 3 months. When Hcy did not fall below 20 mol/L, folate doses were increased by 5 mg every 3 months to up to 15 mg/day. With 5 mg/day supplementation, serum folate concentrations increased above the upper confidence limit in 23 patients and erythrocyte folate concentrations in 27 patients. Hcy levels decreased to less than 15 mol/L in 6 cases and by more than 50% in 12 cases. Nineteen of the remaining patients were given 10 mg/day folate. After increasing the dose, serum and erythrocyte folate levels rose above the upper detection limit. In one patient, plasma Hcy concentrations decreased to less than 15 mol/L. Ten patients were given 15 mg/day oral folate for an additional 3 months with no effect on homocystinemia. This study confirms that oral folate supplementation may improve hyperhomocystinemia even in patients on dialysis with normal serum or erythrocyte folate concentrations. In fact, serum and erythrocyte levels cannot predict the effect of supplementation on plasma Hcy levels. However, 5 mg/day folate supplementation normalized Hcy in 10% of cases and reduced Hcy levels in another 21%. Increasing the folate dose to greater than 5 mg/day had a minimal (10 mg/day) or no (15 mg/day) additional effect on Hcy concentrations. Despite the minimal effect of increasing folate doses, given the low cost, the absence of side effects, and the high cardiovascular risk for patients on peritoneal dialysis, a careful attempt to increase the dose of oral folate up to 10 mg/day might be suggested

Glutamate-cysteine ligase polymorphism, hypertension and male gender are associated with cardiovascular events : biochemical and genetic characterization of an Italian sub-population

Background: Glutathione (GSH) is an important intravascular scavenger that protects endothelial cells from atherosclerosis. However, it is still unknown whether cardiovascular (CV) events are associated with metabolic and genetic factors, linked to GSH synthesis in an Italian subpopulation, and if a glutamate-cysteine ligase polymorphism within the catalytic subunit (GCLC) could affect blood and plasma GSH concentrations. Methods: One hundred subjects, with or without CV risk factors, were enrolled to evaluate plasma and erythrocyte redox status (GSH, homocysteine, cysteine, cysteinylglycine), antioxidant vitamins (\u3b1-tocopherol and ascorbate), malondialdehyde, a lipid peroxidation product, and the presence of the GCLC-129 C/T polymorphism; an experimental hyperhomocysteinemia after methionine-induced stimulation of transsulfuration pathway was performed in 91% of enrolled subjects. Clinical, biochemical, and genetic variables were correlated with the presence of CV events (myocardial infarction, transient ischemic attacks, and stroke). Results: By multiple logistic regression analysis, male sex (P = .027), hypertension (P = .001), and GCLC C/T genotype (P = .009) were the only variables associated with events. Plasma \u3b1-tocopherol content decreased postmethionine in the T allele subjects compared with wild type (P for time 7 group interaction = .001). Plasma-reduced GSH level was higher in C/T than in C/C genotype subjects at both time points (P for group = .03), whereas intracellular GSH concentration did not differ between the 2 genotype groups either at baseline or postmethionine. Conclusions: GCLC T allele, together with hypertension and male sex, is associated with CV events in our study population. Moreover, after stimulation of transsulfuration, intracellular GSH content is preserved in T allele subjects, probably by increases in GSH turnover and export, and consumption of \u3b1-tocopherol