Association between carrier screening and incidence of cystic fibrosis

Context A downward trend in cystic fibrosis (CF) birth incidence has been reported in some areas. Objective To evaluate the association between carrier screening and CF birth incidence. Design, Setting, and Participants In northeastern Italy, CF birth incidence is monitored by means of a long-standing neonatal screening program. In the same area, 2 sections using different carrier detection approaches were identified—the western region, in which CF carrier tests are offered only to relatives of patients or to couples planning in vitro fertilization; and the eastern region, in which carrier testing is offered to relatives and carrier screening to infertile couples and to couples of reproductive age. A total of 779 631 newborns underwent CF neonatal screening between January 1993 and December 2007, of whom 195 had CF detected. Main Outcome Measure Cystic fibrosis birth incidence in the 2 regions. Results A time-related decrease in birth incidence was found, with a mean annual percentage decrease of 0.16 per 10 000 neonates (P < .001). In the western region, 2559 carrier tests were performed, 314 carriers were identified, and 9 carrier couples were detected. In the eastern region, 87 025 carrier tests were performed, 3650 carriers were identified, and 82 carrier couples were detected. The birth rate decrease was greater in the eastern region (decrease rate, 0.24; 95% confidence interval [CI], 0.12-0.36) than in the western region (decrease rate, 0.04; 95% CI, –0.16 to 0.08; P = .01). The increase in the number of screened carriers over time was significantly correlated with the decrease in CF birth incidence (correlation coefficient = −0.53; 95% CI, –0.20 to –0.74; P = .003). Conclusion In northeastern Italy, carrier screening was associated with a decrease in the incidence of CF

Impaired CFTR function in mild cystic fibrosis associated with the S977F/T5TG12complex allele in trans with F508del mutation

AbstractBackgroundThe S977F mutation (c.2930C>T) in the CFTR gene (CFTR/ABCC7) is extremely rare. We describe the case of an adult patient carrying the complex allele S977F/T5TG12 in trans with the F508del mutation. Mild respiratory manifestations arose in adulthood associated with azoospermia, acute pancreatitis, minor hemoptysis and Cl− levels ranging from 40 to 42mEq/L.MethodDiagnosis was confirmed by repeated NPD measurements, genetic DHPLC analysis and a recently described functional assay measuring cAMP-dependent cell depolarization in peripheral blood monocytes.ResultsNPD measurements, DHPLC and monocyte functional assay (CF index=−18). Results were consistent with a CF phenotype.ConclusionsThe combined application of DHPLC and NPD analysis in the algorithm for CF diagnosis appears useful for the management of similar cases. In addition, the novel monocyte functional assay might contribute to improve our diagnostic capability, counseling and better treatment of these challenging clinical cases

Challenging the diagnosis of Cystic Fibrosis in a patient carrying the 186-8T/C allelic variant in the CF Transmembrane Conductance Regulator gene

BACKGROUND: This report describe for the first time a clinical case with a CFTR allelic variant 186-8T/C (c.54-8 T/C) in intron 1 of CFTR and underline the importance of applying a combination of genetic and functional tests to establish or exclude a diagnosis of Cystic Fibrosis. In this case the diagnostic algorithm proposed for CF has been successfully applied at our Center and previous CF diagnosis assigned in a different Center was not confirmed.Case report: A 38 year-old Italian woman had been treated as affected by CF since 2010, following diagnosis based on sweat tests (reported values of 73 and 57 mEq/L) and a clinical history consistent with CF. No mutations were identified by first level of genetic analysis. Afterwards the patient referred to our center for assessing the relevance of these findings. The genetic variant 186-8T/C (c.54-8 T/C) in intron 1 of the CFTR gene was detected by sequencing. Low-level interstitial-alveolar infiltration was recorded by high-resolution computerized tomography. Lung function was normal and sputum and Broncho Alveolar Lavage cultures resulted bacteriologically negative. Sweat chloride levels was re-assessed and resulted with values of 57 and 35 mEq/L, with a borderline range between 40 and 60 mEq/L. Nasal Potential Difference measurements resulted in three reliable measurements consistent with a non-CF phenotype. Differential diagnosis with ciliary dyskinesia was excluded, as was exon 2 skipping of CFTR gene that might have caused a CFTR functional defect. Furthermore, single cell fluorescence analysis in response to cAMP agonists performed in patient's monocytes overlapped those obtained in healthy donors. CONCLUSION: We concluded that this patient was not affected by CF. This case highlights the need for referrals to highly specialized centers and the importance of combined functional and genetic tests in making a correct diagnosis. Moreover, we confirmed a correlation between NPD tracings and cell depolarization in monocytes providing a rationale for proposing the use of leukocytes as a potential support for CF diagnosis

Defective CFTR Expression and Function Are Detectable in Blood Monocytes: Development of a New Blood Test for Cystic Fibrosis

BACKGROUND: Evaluation of cystic fibrosis transmembrane conductance regulator (CFTR) functional activity to assess new therapies and define diagnosis of cystic fibrosis (CF) is cumbersome. It is known that leukocytes express detectable levels of CFTR but the molecule has not been characterized in these cells. In this study we aim at setting up and validating a blood test to evaluate CFTR expression and function in leukocytes. DESCRIPTION: Western blot, PCR, immunofluorescence and cell membrane depolarization analysis by single-cell fluorescence imaging, using the potential-sensitive DiSBAC(2)(3) probe were utilized. Expression of PKA phosphorylated, cell membrane-localized CFTR was detected in non-CF monocytes, being undetectable or present in truncated form in monocytes derived from CF patients presenting with nonsense mutations. CFTR agonist administration induced membrane depolarization in monocytes isolated from non-CF donors (31 subjects) and, to a lesser extent, obligate CFTR heterozygous carriers (HTZ: 15 subjects), but it failed in monocytes from CF patients (44 subjects). We propose an index, which values in CF patients are significantly (p<0.001) lower than in the other two groups. Nasal Potential Difference, measured in selected subjects had concordant results with monocytes assay (Kappa statistic 0.93, 95%CI: 0.80-1.00). RESULTS AND SIGNIFICANCE: CFTR is detectable and is functional in human monocytes. We also showed that CFTR-associated activity can be evaluated in 5 ml of peripheral blood and devise an index potentially applicable for diagnostic purposes and both basic and translational research: from drug development to evaluation of functional outcomes in clinical trials

Azithromycin Selectively Reduces Tumor Necrosis Factor Alpha Levels in Cystic Fibrosis Airway Epithelial Cells

Azithromycin (AZM) ameliorates lung function in cystic fibrosis (CF) patients. This macrolide has been suggested to have anti-inflammatory properties as well as other effects potentially relevant for therapy of CF. In this study, we utilized three CF (IB3-1, 16HBE14o- AS3, and 2CFSMEo-) and two isogenic non-CF (C38 and 16HBE14o- S1) airway epithelial cell lines to investigate whether AZM could reduce tumor necrosis factor alpha (TNF-α) mRNA and protein levels by real-time quantitative PCR analysis and an enzyme-linked immunosorbent assay (ELISA), respectively. We studied the effects on the DNA binding of NF-κB and specificity protein 1 (Sp1) by an ELISA. Non-CF cells express significantly lower TNF-α mRNA and protein levels than an isogenic CF cell line. In CF cells, AZM treatment causes a 30% reduction of TNF-α mRNA levels (P < 0.05) and a 45% decrease in TNF-α secretion (P < 0.05), reaching approximately the levels of the untreated isogenic non-CF cells. In CF cells, NF-κB and Sp1 DNA binding activities were also significantly decreased (about 45 and 60%, respectively; P < 0.05) after AZM treatment. Josamycin, a macrolide lacking clinically described anti-inflammatory effects, was ineffective. Finally, AZM did not alter the mRNA expression levels of interleukin-6, a proinflammatory molecule not differentially expressed in CF and isogenic non-CF cells. The results of our study support the anti-inflammatory activities of this macrolide, since we show that AZM reduced the levels of TNF-α and propose inhibitions of NF-κB and Sp1 DNA binding as possible mechanisms of this effect

Association between carrier screening and incidence of cystic fibrosis

Context A downward trend in cystic fibrosis (CF) birth incidence has been reported in some areas. Objective To evaluate the association between carrier screening and CF birth incidence. Design, Setting, and Participants In northeastern Italy, CF birth incidence is monitored by means of a long-standing neonatal screening program. In the same area, 2 sections using different carrier detection approaches were identified-the western region, in which CF carrier tests are offered only to relatives of patients or to couples planning in vitro fertilization; and the eastern region, in which carrier testing is offered to relatives and carrier screening to infertile couples and to couples of reproductive age. A total of 779 631 newborns underwent CF neonatal screening between January 1993 and December 2007, of whom 195 had CF detected. Main Outcome Measure Cystic fibrosis birth incidence in the 2 regions. Results A time-related decrease in birth incidence was found, with a mean annual percentage decrease of 0.16 per 10 000 neonates (P < .001). In the western region, 2559 carrier tests were performed, 314 carriers were identified, and 9 carrier couples were detected. In the eastern region, 87 025 carrier tests were performed, 3650 carriers were identified, and 82 carrier couples were detected. The birth rate decrease was greater in the eastern region (decrease rate, 0.24; 95% confidence interval [CI], 0.12-0.36) than in the western region (decrease rate, 0.04; 95% CI, -0.16 to 0.08; P = .01). The increase in the number of screened carriers over time was significantly correlated with the decrease in CF birth incidence (correlation coefficient=-0.53; 95% CI, -0.20 to -0.74; P = .003). Conclusion In northeastern Italy, carrier screening was associated with a decrease in the incidence of CF

New insights into the Shwachman-Diamond Syndrome-related haematological disorder: Hyper-activation of mTOR and STAT3 in leukocytes

Shwachman-Diamond syndrome (SDS) is an inherited disease caused by mutations of a gene encoding for SBDS protein. So far little is known about SBDS exact function. SDS patients present several hematological disorders, including neutropenia and myelodysplastic syndrome (MDS), with increased risk of leukemic evolution. So far, the molecular mechanisms that underlie neutropenia, MDS and AML in SDS patients have been poorly investigated. STAT3 is a key regulator of several cellular processes including survival, differentiation and malignant transformation. Moreover, STAT3 has been reported to regulate neutrophil granulogenesis and to induce several kinds of leukemia and lymphoma. STAT3 activation is known to be regulated by mTOR, which in turn plays an important role in cellular growth and tumorigenesis. Here we show for the first time, to the best of our knowledge, that both EBV-immortalized B cells and primary leukocytes obtained from SDS patients present a constitutive hyper-activation of mTOR and STAT3 pathways. Interestingly, loss of SBDS expression is associated with this process. Importantly, rapamycin, a well-known mTOR inhibitor, is able to reduce STAT3 phosphorylation to basal levels in our experimental model. A novel therapeutic hypothesis targeting mTOR/STAT3 should represent a significant step forward into the SDS clinical practice