17q12 microduplications: a challenge for clinicians.

In the recent years, some cases of 17q12 deletions and duplications have been reported, but the clinical impact of these imbalances is still to be fully elucidated. In particular, 17q12 duplications elude syndrome classification, since they are associated with a wide phenotypic spectrum, ranging from very mild to quite severe phenotypes. Here, two unrelated patients with the same 1.2 Mb microduplication of 17q12 are reported. Comparing these patients' phenotype with those previously published, it emerges that the more patients reported, the more difficult is finding common characteristics, even in presence of exactly the same genetic anomaly. The role of the genes duplicated in this region and the impact of this chromosomal imbalance are discussed.

Myocardial stunning after resuscitation from cardiac arrest following spinal anaesthesia.

Cardiac arrest associated with spinal anaesthesia has been well researched. Myocardial stunning after successful resuscitation from cardiac arrest is seen in up to 2/3(rd) of in-hospital cardiac arrests. Myocardial stunning after resuscitation from cardiac arrest associated with spinal anaesthesia has probably not been reported earlier. Our case, an ASA physical status I lady, posted for tubal reanastomosis surgery developed bradycardia followed by asystole, approximately 5 minutes after giving subarachnoid block. Return of spontaneous circulation (ROSC) was achieved within 2 minutes with cardiopulmonary resuscitation (CPR) and defibrillation for pulseless ventricular tachycardia. Patient developed delayed pulmonary oedema, which was probably due to myocardial stunning. In the present case, inadequate preloading could have precipitated bradycardia progressing to cardiac arrest which, after resuscitation led to reversible myocardial dysfunction. We conclude that early vasopressor infusion, titrated fluids and echocardiography should be considered in immediate post cardiac arrest phase following spinal anaesthesia.

FOLFOXIRI plus bevacizumab as first-line treatment in BRAF mutant metastatic colorectal cancer.

BACKGROUND: BRAF V600E mutation plays a negative prognostic role in metastatic colorectal cancer (mCRC), leading to a median Progression Free Survival (PFS) of 4-6months with first-line conventional treatments. Our group recently reported in a retrospective exploratory analysis of a phase II trial that FOLFOXIRI (5-FU/LV+Oxaliplatin+Irinotecan) plus bevacizumab might allow to achieve remarkable results in terms of PFS and Overall Survival (OS) also in this poor-prognosis subgroup. The aim of this work was to prospectively validate our retrospective finding. PATIENTS AND METHODS: This phase II trial was designed to detect an increase in 6month-Progression Free Rate (6m-PFR) from 45% to 80% in a population of BRAF mutant mCRC patients treated with first-line FOLFOXIRI plus bevacizumab. Secondary end-points were PFS, OS, response rate (RR) and the analysis of outcome parameters in the pooled population consisting of both retrospectively and prospectively included patients. This trial is registered with ClinicalTrials.gov, number NCT01437618. RESULTS: Two-hundred-fourteen potentially eligible mCRC patients were screened for BRAF mutational status. Fifteen BRAF mutant patients (7%) were included in the validation cohort. At a median follow up of 25.7months, 6m-PFR was 73%. Median PFS and OS were 9.2 and 24.1months, respectively. In the pooled population, at a median follow up of 40.4months, 6m-PFR was 84%. Median PFS and OS were 11.8 and 24.1months, respectively. Overall RR and disease control rate were 72% and 88%, respectively. CONCLUSION: Lacking randomised trials in this specific molecular subgroup, FOLFOXIRI plus bevacizumab might be a reasonable option for the first-line treatment of BRAF mutant mCRC patients.

Marine yeast Candida aquaetextoris S527 as a potential immunostimulant in black tiger shrimp Penaeus monodon.

A marine yeast Candida aquaetextoris S527 as a source of immunostimulant in Penaeus monodon was studied. Yeast diet was prepared by incorporating 10% C. aquaetextoris S527 biomass into a standard shrimp diet and administered in P. monodon at different frequencies (daily, once in three days, once in seven days and once in ten days) followed by challenge with white spot syndrome virus (WSSV). Immune parameters such as total protein, total hemocyte count, pro-phenoloxidase, nitroblue tetrazolium reduction, alkaline phosphatase activity and acid phosphatase activity were tested. Expression profile of antimicrobial peptide (AMP) genes viz., anti-lipopolysaccharide factor (ALF), crustin-1, crustin-2, crustin-3, penaeidin-3 and penaeidin-5; immune genes viz., alpha-2-macroglobulin (α-2-M), astakine, peroxinectin, prophenol oxidase (proPO) and transglutaminase, and WSSV genes viz., DNA polymerase, endonuclease, protein kinase, immediate early gene, latency related gene, ribonucleotide reductase, thymidine kinase and VP28 were analyzed. The study demonstrated that marine yeast diet administered once every seven days conferred better protection to P. monodon against WSSV infection, supported by the hematological and immune gene expression profiles analyzed.

Nasal nitric oxide and nitric oxide synthase expression in primary ciliary dyskinesia.

No study has evaluated the correlation between different expression of nitric oxide synthase (NOS) isoforms in nasal epithelial cells and nasal NO (nNO) level in primary ciliary dyskinesia (PCD). Gene expression of endothelial (NOS3) and inducible NOS (NOS2) and their correlation with nNO level, ciliary function and morphology were studied in patients with PCD or secondary ciliary dyskinesia (SCD). NOS3 gene polymorphisms were studied in blood leukocytes. A total of 212 subjects were studied (48 with PCD, 161 with SCD and three normal subjects). nNO level correlated with mean ciliary beat frequency (p = 0.044; r = 0.174). The lower the nNO level the higher was the percentage of immotile cilia (p<0.001; r = -0.375). A significant positive correlation between NOS2 gene expression and nNO levels was demonstrated in all children (p = 0.001; r = 0.428), and this correlation was confirmed in patients with PCD (p = 0.019; r = 0.484). NOS2 gene expression was lower in PCD than in SCD (p = 0.04). The NOS3 isoform correlated with missing central microtubules (p = 0.048; r = 0.447). nNO levels were higher in PCD subjects with the NOS3 thymidine 894 mutation, and this was associated with a higher ciliary beat frequency (p = 0.045). These results demonstrate a relationship between nNO level, NOS mRNA expression and ciliary beat frequency.

Clinical and genetic findings in a series of Italian children with pure hereditary spastic paraplegia.

BACKGROUND: hereditary spastic paraplegias (HSP) are a group of neurodegenerative disorders characterized by progressive lower extremity spastic weakness. SPG7, SPG4 and SPG3A are some of the autosomal genes recently found as mutated in recessive or dominant forms of HSP in childhood. SPG31 is more often associated with a pure spastic paraplegia phenotype, but genotype-phenotype correlation is still unclear. The aims of the current study was: (i) to verify the mutational frequency of SPG4, SPG3A, SPG31 and SPG7 genes in our very-well-selected childhood sample, and (ii) to improve our knowledge about the clinical and electrophysiological HSP phenotypes and their possible correlation with a specific mutation. METHODS: a sample of 14 Italian children affected by pure HSP (mean age at diagnosis 5.9 years) was extensively investigated with electrophysiological, neuroradiological and genetic tests. RESULTS: three SPG4 mutations were identified in three patients: two novel missense mutations, both sporadic, and one multiexonic deletion already reported. A novel large deletion in SPG31 gene involving exons 2-5 was also detected in one young patient. No mutations in the SPG7 and in the SPG3A genes were found. CONCLUSIONS: our data confirm that HSP represent a heterogeneous group of genetic neurodegenerative disorders, also in sporadic or autosomal recessive early onset forms. Multiplex Ligation-dependent Probe Amplification-based mutation screening for SPG4 and SPG31 genes would be added to sequencing-based screening of SPG4, SPG31 and SPG3A genes in the routine diagnosis of HSP children.

Increasing financial burden of revision total knee arthroplasty.

We reviewed the peri-operative and financial data of patients who underwent revision total knee arthroplasty in our institution between 1997 and 2006. The aims were to compare difference in cost between aseptic and septic cases and to identify the sources of preventable cost increase in revision knee procedure. The study group comprised 117 women (65%) and 62 men (35%). The median age of patients decreased from 73 years (37-83 years) in 1997-2001 to 70 years (15-91 years) in 2002-2006, a decline of 4% (P < 0.05). The mean ASA scores also dropped from 3 to 2 between the two periods. Despite this, the mean total cost of revision knee procedure continued to increase. Patients undergoing revision arthroplasty because of infection had much higher (P = 0.0001) cost compared to their aseptic counterpart. Increase in the costs of investigations (P < 0.05) and implant (P < 0.05) was the major contributing factors. The cost of implants increased by 32-35% (P < 0.05) depending on implant selection. Changing demographics will increase the requirement for this surgery and thus increase its overall cost to society. Cost increases associated with unnecessary investigations, prolonged hospital stay and use of expensive implants should be avoided.

A girl with tomboy behavior: lesson from misdiagnosis in a baby with ambiguous genitalia.

5alpha-Reductase-2 deficiency is a rare 46,XY disorder of sex differentiation caused by mutations in the 5alpha-reductase type 2 gene. It presents at birth with variable degree of undervirilization. Here, a baby with 5alpha-reductase-2 deficiency and misdiagnosis of complete androgen insensitivity syndrome, female sex assignment and early gonadectomy is described. During primary school, the girl developed tomboy behavior. Molecular analysis demonstrated compound heterozygosity for 5alpha-reductase type 2 gene mutations (exon 2: Q126R; exon 4: H230P). This child underlines the need for adequate endocrine and genetic testing for a definite diagnosis before gender is assigned in children with ambiguous genitalia and surgical interventions are carried out. Inadequate work-up may result in inappropriate gender assignment in infancy with possible inferences on outcome.

Body composition and metabolic profile in women with complete androgen insensitivity syndrome.

Clinical and experimental data suggest that androgen receptor (AR) signaling plays a role on body composition, glucose homeostasis and lipid metabolism. The effect of AR disruption on such parameters was not extensively investigated in human people. A group of young to middle-age adult women with complete androgen insensitivity syndrome (CAIS, n = 18, age 32.2 +/- 9.3 years; women with testes removed n = 14) was investigated for body mass index (BMI), body composition (dual energy X-ray absorptiometry), serum glucose levels, insulin sensitivity (HOMA-IR) and lipid profile. Mean BMI (24.2 +/- 7.4 kg/m(2)) was not significantly increased (T-score 1.0 +/- 2.5, p = NS vs Italian female reference values), but prevalence of obesity was higher in women with CAIS than that reported in age-related Italian females (16.7% vs 3.6%, respectively). The majority of obese individuals with CAIS was in the subgroup with intact testes (3/4). DXA assessment (n = 15) demonstrated values of total free fat mass similar to that of 46,XX female controls. Increased body fat was found in CAIS women in comparison with both female and male controls. Abnormal values of cholesterol (total and LDL) and HOMA-IR were present in a large subset of patients. Our data suggest that in women with CAIS disruption of AR signaling may increase body fat and affect some metabolic parameters. Assessment of body composition, metabolic profile and, likely, cardiovascular risk seems to be advisable with ageing in these individuals.

Molecular characterization of 6 unrelated Italian patients with 5alpha-reductase type 2 deficiency.

Steroid 5alpha-reductase (5alphaR) deficiency (OMIM number #264600) is a rare 46,XY disorder of sex differentiation caused by mutations in the 5alphaR type 2 gene (SRD5A2) resulting in dihydrotestosterone deficiency during fetal development. We report on the analysis of the SRD5A2 gene in 6 unrelated 46,XY Italian patients with external genitalia morphology ranging from predominantly female to nearly completely male. Three subjects were seen and assessed at birth, 1 patient was referred to us before puberty, and 2 at postpubertal age. Six different causative mutations (5 missense and 1 nonsense) and a rare polymorphism were identified. Four patients presented homozygous single-base substitutions. These SRD5A2 mutations were located in exon 2 (variant Cys133Gly), exon 4 (Gly196Ser and Ala207Asp) and exon 5 (Tyr235Phe). A fifth subject was a compound heterozygote who carried a nonsense mutation in exon 1 (Trp53X) and a second SRD5A2 alteration in exon 5 (Tyr235Phe). The final patient presented a mutation in only 1 allele (Gly34Trp) together with the Ala49Thr variant. The molecular characterization of these patients made it possible to identify novel mutations and to confirm, before gender assignment or any surgical approach, the suspected 5alphaR deficiency in 2 newborns, 1 of whom had inconclusive hormonal data. 5alphaR deficiency in subjects without parental consanguinity and the presence of compound heterozygotic patients suggest that SRD5A2 mutations carrier frequency may be higher than previously thought.

PS-341 (Bortezomib) inhibits proliferation and induces apoptosis of megakaryoblastic MO7-e cells.

PS-341 (Bortezomib) is a dipeptide boronic acid proteasome inhibitor with antitumor activity that induces apoptosis in different human cancer cell lines. We investigated effects of PS-341 (Bortezomib) on cell proliferation, cell cycle progression, induction of apoptosis and differentiation in a megakaryoblastic (MO7-e) cell line. PS-341 was able to retain NF-kappaB in the cytoplasm and inhibit cell growth (IC(50)=22.5 nM), in a dose/time-dependent way. This anti-proliferative activity resulted to be lineage-specific, because other leukemic cell lines (KG1a, K562/R7, HL60/DNR) were unaffected by the PS-341 treatment. Moreover, PS-341 in MO7-e induced a significant pro-apoptotic effect from 10 nM concentration (40% versus 12% in the control, p<0.05). On the other hand, at lower concentration (5 nM), Bortezomib blocked cell cycle in the G2 phase. Finally, this compound was able to down-regulate WT1 expression. No significant effects on cell differentiation were found. Because a spontaneous NF-kappaB activation has been reported in megakaryocytes from patients affected by myeloproliferative disorders, Bortezomib would so be an attractive therapeutic tool for these malignancies, including essential thrombocythemia or idiopathic myelofibrosis. Preliminary data show an inhibiting activity of Bortezomib in the megakaryocytic colonies formation. Finally, also down-regulation of the WT1 gene Bortezomib-driven could be relevant, because of the role that this gene would play in the pathogenesis of acute and chronic myeloproliferative disorders.

Characterization of the phenotype and definition of the deletion in a new patient with ring chromosome 22.

The clinical phenotype of patients with ring chromosome 22 includes mental retardation with severe language impairment, hypotonia, and dysmorphic facial features. In recent years an increasing number of patients with microscopic as well as cryptic terminal deletion involving band 22q13 have been described and their phenotype shows clinical features overlapping with patients with ring chromosome 22. Loss of DNA in the 22q13.3 region may lead to a clinically recognizable syndrome named "22q13.3 deletion syndrome." We report a patient with a ring chromosome 22 who has hypotonia, profound mental retardation, language impairment, dysmorphic features, and behavioral disorders. To check if the critical region responsible for "22q13.3 deletion syndrome" was absent in this ring, a fluorescent in situ hybridization (FISH) analysis using a probe corresponding to the ARSA locus was performed. In our patient, only one ARSA signal could be detected, indicating that the deletion encompassed the critical 22q13.3 region. A more detailed analysis of the deletion extent then was performed using a panel of fluorescent probes located within 22q13. These experiments allowed the identification of the breakpoint between CTA-299D3 and RP5-925J7 probe, located in 22q13.32. Deletion extent could be estimated to be about 2.5 Mb, and this larger deletion may explain the severity of clinical features observed in our patient.

Trisomy 8 in Philadelphia-negative cells during imatinib therapy.

Targeted therapy with imatinib selectively suppresses Philadelphia-positive cells in chronic myeloid leukemia cells, with reappearance of apparently normal hemopoiesis in a considerable number of patients. Recently, clonal abnormalities have been observed in Philadelphia-negative cells during imatinib therapy, the biologic and prognostic significance of which is actually unknown. A case of trisomy 8 occurring in Philadelphia-negative cells, which was treated by bone marrow transplantation, is reported. Chromosomal abnormalities in Philadelphia-negative cells do not seem to herald disease transformation, but the long-term prognosis may be influenced by an increased incidence of myelodysplasia in younger patients.

Nonhomologous Robertsonian translocations (NHRTs) and uniparental disomy (UPD) risk: an Italian multicentric prenatal survey.

OBJECTIVES: The risk of uniparental disomy (UPD) occurrence associated with the prenatal finding of balanced nonhomologous Robertsonian translocations (NHRTs) has been estimated only on limited empirical data. The aim of the study was to verify the estimate of the general risk, to get narrower confidence intervals by cumulating the data and to obtain risk estimates for specific translocation types. METHODS: We tested for UPD 160 prenatal specimens referred to the participant centers after the cytogenetic finding of NHRT. RESULTS: One case of upd(14)mat was found, associated with a 45,XX,der(14;22)mat fetal karyotype. The general empirical risk of UPD occurrence in NHRT carrier fetuses, corrected for the actual number of chromosomes analyzed, was 0.76% (95% CI 0.02-4.25%). Cumulative data with previous studies gives a general risk of UPD associated with NHRT of 0.80% (95% CI 0.17-2.34%). The UPD risk for the specific NHRT der(13;14) did not significantly differ from that of the other NHRTs taken together. CONCLUSION: The present survey confirms the previously estimated risk of occurrence of UPD in offspring of NHRT carriers as a low, but not negligible risk, worth being investigated in prenatal diagnosis.

Identification of Fas (APO-1/CD95) and p53 gene mutations in non-small cell lung cancer.

Fas (APO-1/CD95) is a broadly expressed death receptor involved in a series of physiological and pathological apoptotic processes. One of the possible mechanisms for resistance to apoptosis signaling in the immune system as well as in the pathogenesis of non-lymphoid malignancies is the presence of Fas mutations within the entire gene. We investigated, in 79 non-small cell lung cancer (NSCLC) samples, the promoter and the entire coding region of the Fas gene by polymerase chain reaction, single strand conformation polymorphism and DNA sequencing in order to detect putative alterations. Sixteen of 79 tumor samples (20.2%) were found to have Fas alterations, either in promoter or exon region. Since the loss of Fas apoptotic function might be linked to p53 alterations, which are often involved in the development of NSCLC, we analyzed p53 status in 40 of the 79 NSCLC samples. p53 mutations were found to be more frequently present than Fas gene alterations (25 out of 40 cases, 62.5%). These data increase the knowledge regarding mutations of apoptosis-genes involved in the pathogenesis of NSCLC, and give benefits for the clinical management of this type of tumor.