ABSTRACT
Primary hyperoxaluria type 1 is an autosomal recessive disorder of glyoxylate metabolism, caused by a deficiency of alanine:glyoxylate aminotransferase, which is encoded by a single copy gene (AGXT. The aim of this research was to standardize denaturing high-performance liquid chromatography, a new, sensitive, relatively inexpensive, and automated technique, for the detection of AGXT mutation. Denaturing high-performance liquid chromatography was used to analyze in blind the AGXT gene in 20 unrelated Italian patients with primary hyperoxaluria type I previously studied by other standard methods (single-strand conformation polymorphism analysis and direct sequencing) and 50 controls. Denaturing high-performance liquid chromatography allowed us to identify 13 mutations and the polymorphism at position 154 in exon I of the AGXT gene. Hence the method is more sensitive and less time consuming than single-strand conformation polymorphism analysis for the detection of AGXT mutations, thus representing a useful and reliable tool for detecting the mutations responsible for primary hyperoxaluria type 1. The new technology could also be helpful in the search for healthy carriers of AGXT mutations amongst family members and their partners, and for screening of AGXT polymorphisms in patients with nephrolithiasis and healthy populations.
Subject(s)
Chromatography, High Pressure Liquid/methods , DNA Mutational Analysis/methods , Hyperoxaluria, Primary/diagnosis , Transaminases/genetics , Adolescent , Adult , Child , Child, Preschool , Female , Humans , Infant , Male , Middle Aged , Mutation , Polymerase Chain Reaction , Sensitivity and SpecificityABSTRACT
The aim of the present study was to analyse the in vitro proliferation and cytokine production by alloantigen-stimulated peripheral blood mononuclear cells (PBMC) obtained from patients affected by systemic sclerosis (SSc) and patients with Raynaud's phenomenon (RP). In SSc patients the proliferation of PBMC stimulated in vitro with alloantigens was significantly increased compared with healthy subjects, while no differences were observed for RP patients. Lymphocytes from SSc patients also produced larger amounts of IFN-gamma compared with healthy controls. However, patients with clinically active disease had lower IFN-gamma levels than those found in clinically stable patients. Patients affected by RP showed significantly higher levels of IFN-gamma than healthy subjects. Analysis at the clonal level of the lymphocyte subsets involved in alloantigen stimulation in one patient affected by active SSc, and one subject with RP confirmed the results obtained using PBMC. In particular, in the RP patient but not in the SSc patient, we observed a population of CD4+ T cells which proliferated to alloantigens in vitro and produced high levels of IFN-gamma. We suggest that T lymphocytes producing high levels of IFN-gamma might play a protective role in RP patients and in established scleroderma.
Subject(s)
Interferon-gamma/biosynthesis , Isoantigens/immunology , Raynaud Disease/immunology , Scleroderma, Systemic/immunology , T-Lymphocytes/immunology , Adult , CD4-Positive T-Lymphocytes/immunology , Clone Cells , Female , Humans , Interferon-gamma/blood , Lymphocyte Activation , Lymphocyte Subsets/immunology , Male , Middle Aged , Raynaud Disease/blood , Scleroderma, Systemic/bloodABSTRACT
Coeliac disease (CD) is a multigenic and multifactorial enteropathy triggered by gluten-composing proteins. A possible involvement of the intestinal Aminopeptidase N (APN) was investigated by an association analysis. SSCP analysis detected four variants at position 281, 378, 956 and 2957 (referred to no. g178535, GenBank) that were studied in 193 Italian CD families. The haplotypic combinations were determined from family segregation and pairwise linkage disequilibria (D' = D/Dmax) between the polymorphic sites were calculated. Significant D' values ranged between 0.78 and 0.31. Association with CD was tested by TDT (Transmission Disequilibrium Test) utilizing as markers the nucleotide substitutions and their haplotypic combinations. No statistically significant transmission distortion to the probands or to their clinically silent sibs was observed. Our data exclude an involvement in CD of the tested markers and of further undetected variation in strong linkage disequilibrium (D' approximately equal to 1) with them. The power of the test was not adequate to detect an association with an unknown polymorphism which is not in complete linkage disequilibrium with those analysed.
