CF is one of the most common life-threatening autosomal recessive disorders among Caucasians. To provide an useful CF test and improve the detection rate of CF mutation in the general population, the selection of a mutation panel should be considered for covering the population disease risk. The aim of this study was to evaluate and to compare the performance of 3 different analytical CF molecular assays: INNO-LiPA, NanoChip CF70 and xTAG Cystic Fibrosis. All 3 mutation panels showed a good detection rate in our geographical area. We analyzed 100 DNA samples with INNO-LiPA and NanoChip CF70; half of those samples were also analyzed with xTAG Cystic Fibrosis. All tests included the most frequent CF mutations along with Poly-T screening. As some discordant results were found, some samples were also analyzed by CFTR massive parallel sequencing (MPS) with MASTR v2 assay (Multiplicom) run on 454 GS Junior. INNO-LiPA and NanoChip were concordant for 99 out of 100 samples. Only one, carrying the 852del22 mutation, resulted as discordant: MPS confirmed the "wild-type" genotype previously obtained by NanoCHIP. On the contrary, in a sample genotyped by both INNO-LiPA and MPS as compound heterozygote (327226 A/G; 621+3 A/G), NanoChip only detected the 3272-26 A/G mutation. Finally, 47 out of 50 samples were correctly genotyped by xTAG Cystic Fibrosis.

Cystic fibrosis (CF; OMIM number 219700) is an autosomal recessive disease caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene, which results in abnormal viscous mucoid secretions in multiple organs and whose main clinical features are pancreatic insufficiency, chronic endobronchial infection, and male infertility. We report the case of a 47-year-old apparently normal male resulting in homozygosity for the mutation p.M348K from nonconsanguineous parents. The proband was screened using a standard panel of 70 different tested on NanoChip 400 platform. The massive parallel pyrosequencing on 454 JS machine allowed the second level analysis. The patient was firstly screened with two different platforms available in our laboratory, obtaining an ambiguous signal for the p.R347P mutation. For this reason we decided to clarify the discordant result of CFTR status by Next Generation Sequencing (NGS) using 454 Junior instrument. The patient is resulted no carrier of the p.R347P mutation, but NGS highlighted a homozygous substitution from T>A at position 1043 in the coding region, causing an amino acid substitution from methionine to lysine (p.M348K). Casual finding of p.M348K homozygote mutation in an individual, without any feature of classical or nonclassical CF form, allowed us to confirm that p.M348K is a benign rare polymorphism.

Introduction: Aim of this study is to evaluate the diagnostic performance of PCA3 in patients with indication to perform a new biopsy, according to the histological doubt such as High Grade Prostatic Intraepithelial Neoplasia (HGPIN) or Atypical Small Gland Proliferation (ASAP) or the clinical suspicion. Materials and Methods: One hundred men were enrolled. We used the PCA3 - PROGENSA™ procedure. After the PCA3 test a repeated prostate biopsy was proposed. The histological findings were correlated to the PCA3 scores. We calculated the positive predictive value (PPV), the sensibility, the specificity, the Youden's index, the ROC curves, the area under the curve (AUC) for each cut-off value of PCA3 score. Results: These results are preliminary, because at present only 50 of the 100 enlisted men were subjected to rebiopsy. We calculated the best cut-off PCA3 score 20 at the first diagnosis; for patients with HGPIN or ASAP at first biopsy the best sensitivity cut-off is 45; the best cutoff is 45 when you already have a diagnosis of HGPIN, and 35 for ASAP. If we normalize the PCA3 score to the prostate volume, the best cut-off would be 20, with 100% sensitivity with a prostate volume of 65 ml. All results are statistically significant. The real problem, also present in literature, is the constant presence of not diagnosed prostate cancers, for any cut-off value. Conclusions: Our preliminary results suggest that, to get the best diagnostic performance, it would be wrong to maintain a single cut-off, but it should be chosen according to the scenario of the patients subgroup. It is to explore the possibility to search for the PCA3 in the serum to bridge the gap of the aggressive PCa missed by the urinary test.

Background: The role of PCa3 score in the diagnostics of prostate cancer (PCa) is still under debate, mainly due to the lack of a univocal cut-off useful alone or within nomograms proposed by Urologists. Aim of present study is to compare different PCA3 score cut-off values (20, 25, 35 and 50) observed in 734 patients with suspected PCa who were monitored for about three years with single or multiple biopsies.Methods: 734 patients who underwent first prostate biopsy for suspected PCa were enrolled. One month later the first biopsy result was obtained, both negative and positive PCa patients were investigated by means of PCA3 score, in order to establish risk of PCa presence on repeated biopsies.Results: PCA3 score was significantly higher (p < 0.001) in PCa patients to the PCa negative ones, while tPSA did not significantly vary. The best negative predictive value (NPV 97.5%) and sensitivity (95.4%) result were obtained when a PCA3 score of 20 was used. At cut-off value of 50, the 75% of patients resulted as false positive.Conclusions: PCA3 score of 20 could be safely introduced in the prostate cancer screening diagnostic flow chart, since it provides important information regarding the outcome of re-biopsy. (C) 2013 Elsevier B.V. All rights reserved.

Objective: Mannosebinding lectin (MBL2) is a collectin molecule able to activate the complement system and the subsequent inflammatory mechanisms. Several MBL2 genetic variants have been described, including the six variants studied in this report, which are those analyzed in most detail in the medical literature. Design: The present study analyzes the prevalence of MBL2 gene variants in preterm newborns and associates individual genotypes with pulmonary outcome variables. All polymorphisms were analyzed by means of a commercially available reverse dot - blot kit. Setting: Tertiary neonatal intensive care unit. Patients and participants: Seventy- five consecutive preterm newborns. Measurements and results: Two variants were particularly analyzed: - 550G > C and R52C. The first one is known to be associated with lower protein synthesis when included in specific haplotypes. The homozygous and heterozygous - 550G > C mutations were significantly associated with protective effects regarding different lung outcome variables, including shorter duration of mechanical ventilation, hours of continuous positive airway pressure and lower number of hemotransfusions. In contrast, the heterozygous R52C mutation was associated with unfavorable outcome, including higher bronchopulmonary dysplasia prevalence. Multivariate logistic regression analysis showed that these associations were independent of gestational age and birth weight. In addition, four groups of patients were defined on the basis of haplotype combinations. Those known to be associated with low serum MBL2 levels were linked to a better outcome in terms of factors such as hours of mechanical ventilation, continuous positive airway pressure, number of hemotransfusions and bronchopulmonary disease development. Conclusions: The four haplotype combination groups may have a potential diagnostic use as opposite risk factors for lung disease of prematurity.

Congenital adrenal hyperplasia (CAH) is a genetic disorder due to 21-hydroxylase deficiency. More than 90% of CAH cases are caused by mutations of CYP21B gene, most of which are the result of microconversion events between the functional gene and its pseudogene. Using a combination of RFLP and direct sequencing analysis, in this paper we describe the genetic study of a Sardinian family carrying I172N mutation in linkage with a SNP namely 1636 Int6. The characterization of new polymorphisms in CYP21B can improve the analysis of segregation of CYP21B mutated alleles especially in genetic familial studies. The analysis of linkage between specific mutations and this SNPs, especially if focused on genetic familial studies, may improve the quality of genetic analysis of 21-hydroxylase deficiency.

Background: The molecular monitoring of circulating tumor cells by reverse transcriptase-PCR (RT-PCR) for patients with melanoma, is still under debate. It may be affected by: a) pre-analytical variability, b) frequency of melanoma-associated gene transcripts and c) the reliability of the methods employed. Pew commercial methods are available for the detection of tyrosinase mRNA in blood.Objective: Comparison between two RT-PCR-nested methods with a third one basest on real-time methodology, for detection and quantitation of tyrosinase transcripts, respectively.Methods: Sixty-two melanoma patients with different AJCC stages and 20 healthy subjects were enrolled. All blood samples were extracted in duplicate with two different methods. Two nested-PCR methods (one commercial and one in house) plus a real time commercial kit were employed.Results: The two nested PCR methods employed were overimposable, specific and sensitive, at least in the stage III, where there was a concordance between sentinel lymph nodes detection and blood tyrosinase positivity. The different extraction methods did not affect the quality of results, while the commercial real-time kit cannot be used.Conclusion: Tyrosinase mRNA detection may be therefore employed to monitor the melanoma patients over time in function of response to therapy. (c) 2005 Elsevier B.V. All rights reserved.

We studied the effects of the therapy with recombinant Granulocyte Colony Stimulating Factor (rhG-CSF) on the IGF-I and beta(2)-microglobulin (B2M) levels in bronchoalveolar lavage fluids (BALF) of six neutropenic preterm infants who developed chronic lung disease (CLD) and we compared the results with eleven non-neutropenic neonates with CLD, and 13 non-neutropenic whitout CLD. With the exception of the first day values, there was a clear similarity between non-neutropenic and neutropenic CLD patients. In contrast, non-neutropenic patients without CLD showed significantly lower values of free IGF-I and B2M. These findings suggest that 1) the mechanisms leading to CLD might be mediated by high levels of IGF-family molecules soon after birth 2) B2M could be a marker of BALF cellularity with potential inflammatory properties 3) G-CSF treatment induces an increased synthesis of IGF-I molecules by cells recruited in the lung, with possible enhancement of the fibrogenic mechanisms.