Correction to: Whole exome sequencing in familial isolated primary hyperparathyroidism.

Unfortunately, the 13th author name has been published incorrectly in the original publication.

Whole exome sequencing in familial isolated primary hyperparathyroidism.

PURPOSE: Familial isolated hyperparathyroidism (FIHP) is a rare inherited disease accounting for 1% of all cases of primary hyperparathyroidism (PHPT). It is genetically heterogeneous being associated with mutations in different genes, including MEN1, CDC73, CASR, and recently GCM2. The aim of the study was to further investigate the molecular pathogenesis in Italian FIHP kindreds. METHODS: We used whole exome sequencing (WES) in the probands of seven unrelated FIHP kindreds. We carried out a separate family-based exome analysis in a large family characterized by the co-occurrence of PHPT with multiple tumors apparently unrelated to the disease. Selected variants were also screened in 18 additional FIHP kindreds. The clinical, biochemical, and pathological characteristics of the families were also investigated. RESULTS: Three different variants in GCM2 gene were found in two families, but only one (p.Tyr394Ser), already been shown to be pathogenic in vitro, segregated with the disease. Six probands carried seven heterozygous missense mutations segregating with the disease in the FAT3, PARK2, HDAC4, ITPR2 and TBCE genes. A genetic variant in the APC gene co-segregating with PHPT (p.Val530Ala) was detected in a family whose affected relatives had additional tumors, including colonic polyposis. CONCLUSION: We confirm the role of GCM2 germline mutations in the pathogenesis of FIHP, although at a lower rate than in the previous WES study. Further studies are needed to establish the prevalence and the role in the predisposition to FIHP of the novel variants in additional genes.

Molecular profiling of microinvasive breast cancer microenvironment progression.

BACKGROUND: Tumors develop by progression through a series of stages. Every cell of the tumor microenvironment is constantly changing in the flow of the cancer progression. It has become clear in recent years that stroma is essential for tumor maintenance and growth. Here, we aimed to give a chronological order of gene expression changes given in the dynamical framework of microinvasive breast cancer microenvironment. METHODS: RNA-seq was performed on seven microinvasive breast cancers. For each of them we microdissected seven different portions of the tumor, four related to the breast epithelium and three to the stroma. Breast epithelium was chronologically subdivided in normal breast epithelium (NBE), carcinoma in situ (CIS), emerging invasive fingers (EIF) and invasive breast cancer (IBC). For each of the breast epithelium subdivisions we collected the adjacent stroma (S): S-NBE, S-EIF and S-IBC. RESULTS: The overall differentially expressed genes (DEGs) in all the compartments were analysed and evaluated to understand the pathways involved in tumor progression. Then we analysed the DEGs of the epithelial and stromal portions in comparison with the normal portions. We observed that the stromal cells are necessary for the development and the maintenance of the tumor, especially in tumor progression. Moreover the most important genes involved in the main metabolic pathways were analysed and the communications within the different cell compartments were highlighted. CONCLUSIONS: As a future perspective, a deeply study of the identified key genes, particularly in the stromal cells, will be crucial to develop an anticancer therapy that is undergoing a conversion from a cancer cell-centric strategy to a stroma-centric strategy, more genomically stable.

Association between RAD 51 rs1801320 and susceptibility to glioblastoma.

Glioblastoma is the most common and aggressive malignant primary brain tumor. Despite decades of research and the advent of new therapies, patients with glioblastoma continue to have a very poor prognosis. Radiation therapy has a major role as adjuvant treatment for glioblastoma following surgical resection. Many studies have shown that polymorphisms of genes involved in pathways of DNA repair may affect the sensitivity of the cells to treatment. Although the role of these polymorphisms has been investigated in relation to response to radiotherapy, their role as predisposing factors to glioblastoma has not been clarified yet. In the present study, we evaluated the association between polymorphisms in DNA repair genes, namely: XRCC1 rs25487, XRCC3 rs861539 and RAD51 rs1801320, with the susceptibility to develop glioblastoma. Eighty-five glioblastoma patients and 70 matched controls were recruited for this study. Data from the 1000 Genomes Project (98 Tuscans) were also downloaded and used for the association analysis. Subjects carrying RAD51 rs1801320 GC genotype showed an increased risk of glioblastoma (GC vs GG, χ(2) = 10.75; OR 3.0087; p = 0.0010). The C allele was also significantly associated to glioblastoma (χ(2) = 8.66; OR 2.5674; p = 0.0032). Moreover, RAD51 rs1801320 C allele increased the risk to develop glioblastoma also when combined to XRCC1 rs25487 G allele and XRCC3 rs861539 C allele (χ(2) = 6.558; p = 0.0053).

Antioxidant and anti-inflammatory effects of quercetin in functional and morphological alterations in streptozotocin-induced diabetic rats.

The aim of this study was to investigate functional and morphological alterations caused by oxidative stress in streptozotocin (STZ)-induced diabetic rats and to evaluate the antioxidant effect of quercetin (QUE) in this disease. One hundred and thirty male Wistar rats, it were randomly distributed in 10 different experimental groups, with ten animals per group: Control Saline (CS), Control Ethanol (CE), Control QUE 5mg/kg (CQ5), Control QUE 25mg/kg (CQ25), Control QUE 50mg/kg (CQ50), Diabetic Saline (DS), Diabetic Ethanol (DE), Diabetic QUE 5mg/kg (DQ5), Diabetic QUE25 mg/kg (DQ25), Diabetic QUE 50mg/kg (DQ50). Therefore, hyperglycemia is directly involved in oxidative stress production, as well as in functional and morphological alterations caused by the excess of free radicals. QUE, specially at the dosage of 50mg/kg, can act as an antioxidant and anti-inflammatory agent, becoming a promising adjuvant in the treatment of diabetes mellitus.

Enzymatic and biochemical characterization of Bungarus sindanus snake venom acetylcholinesterase

This study analyses venom from the elapid krait snake Bungarus sindanus, which contains a high level of acetylcholinesterase (AChE) activity. The enzyme showed optimum activity at alkaline pH (8.5) and 45ºC. Krait venom AChE was inhibited by substrate. Inhibition was significantly reduced by using a high ionic strength buffer; low ionic strength buffer (10 mM PO4 pH 7.5) inhibited the enzyme by 1. 5mM AcSCh, while high ionic strength buffer (62 mM PO4 pH 7.5) inhibited it by 1 mM AcSCh. Venom acetylcholinesterase was also found to be thermally stable at 45ºC; it only lost 5% of its activity after incubation at 45ºC for 40 minutes. The Michaelis-Menten constant (Km) for acetylthiocholine iodide hydrolysis was found to be 0.068 mM. Krait venom acetylcholinesterase was also inhibited by ZnCl2, CdCl2, and HgCl2 in a concentrationdependent manner. Due to the elevated levels of AChE with high catalytic activity and because it is more stable than any other sources, Bungarus sindanus venom is highly valuable for biochemical studies of this enzyme.(AU)

Peroxidação lipídica em ratos tratados com sulfato de vincristina e decanoato de nandrolona / Lipid peroxidation in rats treated with vincristine sulphate and nandrolone decanoate

Brain and serum lipid peroxidation was studied in rats treated with vincristine sulphate and different doses of nandrolone decanoate. Thirty rats were distributed into six groups (n=5). The treatments were applied once a week for two weeks. Sample collection was performed in the third week. Treatments during the first week were: G1 (control) - physiologic solution, G2 - vincristine sulphate (4mg/m²), G3 - physiologic solution, G4 - physiologic solution, G5- vincristine sulphate (4mg/m²), and G6 - vincristine sulphate (4mg/m²). In the second week, they were: G1 (control) - physiologic solution, G2- physiologic solution, G3 - nandrolone decanoate (1.8mg/kg-1), G4 - nandrolone decanoate (10mg/kg-1), G5 - nandrolone decanoate (1.8mg/kg-1), and G6 - nandrolone decanoate (10mg/kg-1). Lipid peroxidation increased with the isolated use of vincristine and nandrolone decanoate, and with vincristine associated to the highest dose of the ester as well. These results suggest that vincristine sulphate and nandrolone decanoate increase free radical production. Therapeutic dose of nandrolone decanoate when associated with vincristine sulphate proved to be beneficial, as it was able to protect the organism from damaging processes involved in free radical production.

Este estudo teve por objetivo detectar a peroxidação lipídica presente no cérebro e no soro de ratos tratados com sulfato de vincristina e diferentes doses de decanoato de nandrolona. Trinta ratos foram distribuídos em seis grupos (n=5). Os tratamentos foram aplicados uma vez por semana, durante duas semanas, e a coleta de amostras foi realizada na terceira semana. Na primeira semana, os tratamentos consistiram de: G1(controle) - solução fisiológica; G2 - sulfato de vincristina (4mg/m 2 ); G3 - solução fisiológica; G4 - solução fisiológica; G5 - sulfato de vincristina (4mg/m 2 ) e G6 - sulfato de vincristina (4mg/m 2 ). Na segunda semana: G1(controle) - solução fisiológica; G2 - solução fisiológica; G3 - decanoato de nandrolona (1.8mg/kg-1 ); G4 - decanoato de nandrolona (10mg/kg-1 ); G5 - decanoato de nandrolona (1.8mg/kg-1 ) e G6 - decanoato de nandrolona (10mg/kg-1 ). A peroxidação lipídica aumentou com o uso isolado tanto da vincristina quanto do decanoato de nandrolona e com a associação da vincristina à dose mais alta do éster. Estes resultados sugerem que o sulfato de vincristina e o decanoato de nandrolona aumentam a produção de radicais livres. A dose terapêutica do decanoato de nandrolona, quando associada ao sulfato de vincristina, provou ser benéfica, já que foi capaz de proteger o organismo dos processos prejudiciais induzidos pela produção de radicais livres.

Activity of ectonucleotidases and adenosine deaminase in rats exposed to cigarette smoke.

Cigarette smoke is a complex mixture of various toxic substances that are capable of initiating oxidative damage and promoting blood platelet alterations. In this study, we investigated the activities of the ectoenzymes NTPDase (ectonucleoside triphosphate diphosphohydrolase, CD39) and 5'-nucleotidase (CD73) in platelets as well as adenosine deaminase (ADA) in the plasma of rats exposed to aged and diluted sidestream smoke during 4 weeks. The rats were divided into two groups: I (control) and II (exposed to smoke). After the exposure period, blood was collected and the platelets and plasma were separated for enzymatic assay. The results demonstrated that NTPDase (with ATP as substrate) and 5'-nucleotidase (AMP as substrate) activities were significantly higher in group II (p < 0.05) as compared to group I, while no significant difference was observed for NTPDase with ADP as substrate. The ADA activity was significantly reduced in group II (p < 0.05) as compared with group I. Platelet aggregation was significantly increased in group II (p < 0.05) as compared with group I. We suggest that these alterations in the activity of enzymes from the purinergic system are associated with an increase in platelet aggregation. However, our study has demonstrated that the organism tries to compensate for this enhanced aggregation by increasing hydrolysis of AMP and reducing hydrolysis of adenosine, a potent inhibitor of aggregation and an important modulator of vascular tone.

Acetylcholinesterase activity in rats experimentally demyelinated with ethidium bromide and treated with interferon beta.

The ethidium bromide (EB) demyelinating model was associated with interferon beta (IFN-beta) to evaluate acetylcholinesterase (AChE) activity in the striatum (ST), hippocampus (HP), cerebral cortex (CC), cerebellum (CB), hypothalamus (HY), pons (PN) and synaptosomes from the CC. Rats were divided into four groups: I control (saline), II (IFN-beta), III (EB) and IV (EB and IFN-beta). After 7, 15 and 30 days rats (n = 6) were sacrificed, and the brain structures were removed for enzymatic assay. AChE activity was found to vary in all the brain structures in accordance with the day studied (7-15-30 days) (P < 0.05). In the group III, there was an inhibition of the AChE activity in the ST, CB, HY, HP and also in synaptosomes of the CC (P < 0.05). It was observed that IFN-beta per se was capable to significantly inhibit (P < 0.05) AChE activity in the ST, HP, HY and synaptosomes of the CC. Our results suggest that one of the mechanisms of action of IFN-beta is through the inhibition of AChE activity, and EB could be considered an inhibitor of AChE activity by interfering with cholinergic neurotransmission in the different brain regions.

Apyrase and 5'-nucleotidase activities in synaptosomes from the cerebral cortex of rats experimentally demyelinated with ethidium bromide and treated with interferon-beta.

Apyrase and 5'-nucleotidase activities were analyzed in an ethidium bromide (EB) demyelinating model associated with interferon-beta (IFN-beta). The animals were divided in groups: I, control (saline); II, saline and IFN-beta; III, EB and IV, EB and IFN-beta. After 7, 15 and 30 days the animals (n = 5) were sacrificed and the cerebral cortex was removed for synaptosome preparation and enzymatic assays. Apyrase activity using ATP as substrate increased in groups II, III and IV (P < 0.001) after 7 days and in groups III and IV (P < 0.001) after 15 days. Using ADP as substrate, an activation of this enzyme was observed in group III (P < 0.05) after seven and 15 days. The 5'-nucleotidase activity increased in group III (P < 0.05) after 7 days and in groups II, III and IV (P < 0.001) after 15 days. After 30 days treatment, no significant alteration was observed in enzyme activities. Results showed that apyrase and 5'-nucleotidase activities are altered in demyelination events and that IFN-beta was able to regulate the adenine nucleotide hydrolysis.

Effect of COMT Val108/158 Met genotype on frontal lobe function and risk for schizophrenia.

Abnormalities of prefrontal cortical function are prominent features of schizophrenia and have been associated with genetic risk, suggesting that susceptibility genes for schizophrenia may impact on the molecular mechanisms of prefrontal function. A potential susceptibility mechanism involves regulation of prefrontal dopamine, which modulates the response of prefrontal neurons during working memory. We examined the relationship of a common functional polymorphism (Val(108/158) Met) in the catechol-O-methyltransferase (COMT) gene, which accounts for a 4-fold variation in enzyme activity and dopamine catabolism, with both prefrontally mediated cognition and prefrontal cortical physiology. In 175 patients with schizophrenia, 219 unaffected siblings, and 55 controls, COMT genotype was related in allele dosage fashion to performance on the Wisconsin Card Sorting Test of executive cognition and explained 4% of variance (P = 0.001) in frequency of perseverative errors. Consistent with other evidence that dopamine enhances prefrontal neuronal function, the load of the low-activity Met allele predicted enhanced cognitive performance. We then examined the effect of COMT genotype on prefrontal physiology during a working memory task in three separate subgroups (n = 11-16) assayed with functional MRI. Met allele load consistently predicted a more efficient physiological response in prefrontal cortex. Finally, in a family-based association analysis of 104 trios, we found a significant increase in transmission of the Val allele to the schizophrenic offspring. These data suggest that the COMT Val allele, because it increases prefrontal dopamine catabolism, impairs prefrontal cognition and physiology, and by this mechanism slightly increases risk for schizophrenia.

Excess tryptophan hydroxylase 17 779C allele in surviving cotwins of monozygotic twin suicide victims.

OBJECTIVE: To evaluate the relationship of the tryptophan hydroxylase (TPH) genotype to suicidality by the study of surviving monozygotic (MZ) cotwins of twins who committed suicide. METHOD: Twenty-four surviving Swedish MZ twins whose MZ cotwins had committed suicide were compared to 158 demographically sampled Swedish general population controls for TPH alleles. We also examined serotonin transporter alleles. RESULTS: The living MZ cotwins of suicide victims had a significantly higher TPH 17 779C allele frequency than controls. No significant difference was observed for serotonin transporter alleles. CONCLUSION: These results, in a small sample, suggest the possibility that the 17 779C allele of the TPH gene may be associated with an increased risk of suicide. Further studies in larger samples are needed.

DNA melting analysis for detection of single nucleotide polymorphisms.

BACKGROUND: Several methods for detection of single nucleotide polymorphisms (SNPs; e.g., denaturing gradient gel electrophoresis and denaturing HPLC) are indirectly based on the principle of differential melting of heteroduplex DNA. We present a method for detecting SNPs that is directly based on this principle. METHODS: We used a double-stranded DNA-specific fluorescent dye, SYBR Green I (SYBR) in an efficient system (PE 7700 Sequence Detector) in which DNA melting was controlled and monitored in a 96-well plate format. We measured the decrease in fluorescence intensity that accompanied DNA duplex denaturation, evaluating the effects of fragment length, dye concentration, DNA concentration, and sequence context using four naturally occurring polymorphisms (three SNPs and a single-base deletion/insertion). RESULTS: DNA melting analysis (DM) was used successfully for variant detection, and we also discovered two previously unknown SNPs by this approach. Concentrations of DNA amplicons were readily monitored by SYBR fluorescence, and DNA amplicon concentrations were highly reproducible, with a CV of 2.6%. We readily detected differences in the melting temperature between homoduplex and heteroduplex fragments 15-167 bp in length and differing by only a single nucleotide substitution. CONCLUSIONS: The efficiency and sensitivity of DMA make it highly suitable for the large-scale detection of sequence variants.

Association between seasonal affective disorder and the 5-HT2A promoter polymorphism, -1438G/A.

Genes involved in serotonin metabolism are good candidates for the pathogenesis of seasonal affective disorder (SAD). A functional variant in the serotonin transporter promoter, 5-HTTLPR, has recently been shown to be associated with SAD and seasonality. The purpose of this study was to determine whether -1438G/A, a polymorphism in the 5-HT2A promoter, is associated with SAD and seasonality, and whether it has additive effects with 5-HTTLPR on seasonality. Sixty-seven individuals with SAD and 69 normal volunteers, all screened with the SCID and diagnosed according to DSM-III-R criteria, were genotyped for the -1 438G/A 5-HT2A promoter polymorphism. All had been previously genotyped for 5-HTTLPR and had been assessed for seasonality by the Global Seasonality Scale. There was a significant increase in the frequency of the -1438A variant allele of the 5-HT2A promoter polymorphism in SAD patients (0.47) compared to matched controls (0.36) (P < 0.01). The difference in genotype distribution was also significant (P < 0.05). We found no association between the -1438G/A polymorphism and seasonality scores, and there was no additive effect with 5-HTTLPR on seasonality. In conclusion, we have shown that the -1438G/A 5-HT2A promoter variant is associated with SAD but not with seasonality. We suggest that the association may instead be with the depressive symptoms of SAD. However, these results should be treated with caution until replicated because of the possibility of false-positive findings in case-control association studies.

Role of the serotonin transporter promoter polymorphism in anxiety-related traits.

BACKGROUND: The heritability of interindividual variation in anxiety and other aspects of personality establishes that variants of genes influence these traits. A functional polymorphism in the promoter of the human serotonin transporter gene (SLC6A4*C) was identified and found to be linked to an anxiety-related personality trait, Neuroticism. The polymorphism affects gene transcription and, ultimately, gene function. We have attempted to confirm the role of SLC6A4*C in anxiety-related personality traits by sibpair analysis and association studies. METHODS: Sibpair linkage analysis and association study were performed in 655 Finns. The index cases were 182 alcoholic criminal offenders, through which 258 relatives were ascertained to obtain 366 sibpairs. In addition, 215 unrelated population controls were collected. Each individual was psychiatrically interviewed, blind-rated for DSM-III-R diagnoses, and assessed with the Tridimensional Personality Questionnaire. RESULTS: The sibpair analysis revealed a positive linkage between SLC6A4*C and the 2 anxiety-related subdimensions of Harm Avoidance: HA1 (Anticipatory Worry) and HA2 (Fear of Uncertainty) (P = .003). However, there was no consistent association between SLC6A4*C and any Tridimensional Personality Questionnaire trait. CONCLUSIONS: In the present study we replicated the relationship of SLC6A4*C to anxiety by sibpair linkage analysis but found no evidence of association, raising the question of whether SLC6A4*C locus is itself affecting anxiety or is linked to another still unknown functional variant.

Role of serotonin transporter promoter repeat length polymorphism (5-HTTLPR) in seasonality and seasonal affective disorder.

Seasonal variations in mood and behavior (seasonality) and seasonal affective disorder (SAD) have been attributed to seasonal fluctuations in brain serotonin (5-HT). the short (s), as opposed to the long (l), allele of the 5-HT transporter linked polymorphism (5-HTTLPR) has been associated with neuroticism and depression. We hypothesized that this short allele would also be associated with SAD and with higher levels of seasonality. Ninety-seven SAD patients and 71 non-seasonal healthy controls with low seasonality levels were genotyped for 5-HTTLPR and compared statistically. Patients with SAD were less likely to have the l/l genotype (27.8% vs 47.9%; P < 0.01) and more likely to have the s allele (44.8% vs 32.4%; P < 0.02) as compared to controls. The three 5-HTTLPR genotypes were also differentially distributed in patients and controls (P < 0.03). The SAD patients with the l/l genotype had a lower mean seasonality score than did patients with the other two genotypes (mean +/- s.d. = 15.3 +/- 2.8 vs 17.1 +/- 3.4 respectively; P < 0.02). The 5-HTTLPR short allele contributes to the trait of seasonality and is a risk factor for SAD, providing further evidence for a relationship between genetic variation in the 5-HT transporter (5-HTT) and behavior.

Identification of a Ser857-Asn857 substitution in DRK1 (KCNB1), population frequencies and lack of association to the low voltage alpha EEG trait.

A nonconservative amino acid substitution (Ser857Asn) in the human delayed-rectifier potassium channel DRK1 (KCNB1 locus), a candidate gene for the low voltage alpha electroencephalogram (EEG) trait locus (LVEEG1) at 20q13.2, and its frequency in ethnic population samples are described. The frequency of Asn857 in seven different ethnic population samples, totalling more than 1600 individuals, ranged from zero to greater than 3%. However, no association was found between Asn857 and the low voltage alpha EEG trait (LVA) in a population of 105 subjects assessed for the EEG, 24 of whom actually had LVA.