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BACKGROUND: More than 90% of the cases of Congenital Adrenal Hyperplasia (CAH) are associated with mutations in 21-hydroxylase gene (CYP21A2). Up to now, large CYP21A2 rearrangements have been mainly detected by Southern blot analysis, although more rapid methods have been alternatively proposed. In this paper, we report the use of a multiplex ligation-dependent probe amplification (MLPA) method for easy and rapid detection of deletions/duplications in the CYP21A2 gene. METHODS: We collected 18 CAH Italian patients previously analyzed by gene sequencing and Southern blot technique. In addition, a prenatal diagnosis study was performed. RESULTS: Of the 7 known subjects with CYP21A2 deletions and 2 with gene duplications previously characterized in our laboratory, all were successfully identified by the MLPA analysis. In the prenatal diagnosis study, the MLPA assay was able to identify the presence of a CYP21A2 gene duplication in the fetus, as well in other two family members. CONCLUSION: MLPA analysis represents a simple, rapid and sensitive tool for the detection of CYP21A2/CYP21A1P deletions/duplications in CAH molecular diagnosis. Compared to Southern blot, MLPA may be considered a high throughput analysis, allowing the simultaneous study of several samples in the same experiment and the investigation of both gene (CYP21A2) and pseudogene (CYP21A1P) in each patient.

Involvement of various gene mutations in hypertension have been already reported, including GST (detoxification Phase II enzymes), with contrasting results. This study analyzes a possible association between GSTM1-null and GSTT1-null polymorphisms and the hypertension status in a very old population (>80 years).

Bronchoalveolar lavage fluid (BALF) is an important diagnostic source to investigate molecular changes occurring in lung disorders. The objective of this study was to assess and compare the peptidomic profiles of BALF from premature neonates with and without bronchopulmonary dysplasia (BPD). Samples were obtained on the 3rd day of life from 34 neonates with gestational age a parts per thousand currency sign32 weeks. Two pools of samples from patients with and without BPD were analyzed by high performance liquid chromatography. Several differentially expressed peptides were collected and sequenced. Moreover, samples from single donors were analyzed by liquid chromatography-electrospray ionization mass spectrometry to define the molecular mass values of various peptides and to quantify their expression. Levels of some matrix metalloproteinases and their tissue inhibitors were also determined in single samples. Neonates of the BPD group (N = 16) showed significantly lower mean gestational age and birth weight with respect to the no-BPD group (N = 18; P < 0.0001). Levels of six peptides were significantly higher in BPD patients (P < 0.05). Two of them were identified as the albumin fragments 1-21 (2,428 Da) and 399-406 (956 Da). Levels of matrix metalloproteinase-3 (MMP-3) enzyme probably involved in albumin fragment generation were also significantly higher in the BPD group compared to the no-BPD group (P < 0.05), whereas the levels of tissue inhibitor of metalloproteinases-1 were significantly lower (P < 0.05). Levels of albumin fragments and MMP-3 showed a significant correlation (P < 0.05). This study shows that proteomic techniques can be applied to investigate the involvement of proteolytic enzymes on the airways of mechanically ventilated premature infants.

Patency of the ductus arteriosus (PDA) and bronchopulmonary dysplasia (BPD) development represent severe affections for premature newborns, therefore the research of early markers for these two conditions is really important. The aim of this study is to analyze epithelial lining fluid (ELF) Neutrophil-gelatinase-associated lipocalin (NGAL) levels for prediction of lung injury or possible involvement of this molecule in PDA. Only scarce and contrasting results have previously been published in this field. In contrast, this molecule, included in a large macromolecular complex together with matrix metalloproteinase-9 (MMP-9), is considered an acceptable marker of infectious/inflammatory processes, cancer monitoring and induction of apoptotic pathway. NGAL was detected in 28 pre-term newborns by means of a commercially available kit in bronchoalveolar lavage fluid (BALF). The results have been corrected to ELF levels, by the urea method, to eliminate bias due to BALF collection. ELF NGAL levels were found significantly increased both in infants developing BPD or in those affected by PDA. By means of multivariate logistic regression analysis the significances were confirmed after adjusting for possible interfering variables such as gestational age and concomitant presence of both PDA and BPD. Our results stress the involvement of NGAL in the mechanisms leading to BPD and also suggest a possible association with PDA, which is often linked to prematurity and BPD development, probably due to the involvement of inflammatory and angiogenetic processes in both pathologies.