Fetal DNA in maternal plasma in twin pregnancies.
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DNA↗
Biomedical subjects
Maurizio Ferrari
Publications and source records attributed to Maurizio Ferrari.
Molecular diagnostics by microelectronic microchips.
Molecular diagnostics is being revolutionized by the completion of the human genome project and by the development of highly advanced technologies for DNA testing. One of the most important challenges is the introduction of high throughput systems such as DNA chips into diagnostic laboratories. DNA microchips are small devices permitting rapid analysis of genetic information, exploiting miniaturization of all components and automation of operational procedures. The most important biochip applications include gene expression and genetic variation identification and both may improve human molecular diagnostics. Here we review several approaches developed to allow rapid detection of many single nucleotide polymorphisms and mutations in large population samples. Among these, the use of microelectronics seems to best fit with the needs of molecular diagnostics.
Gene Expression Profiling↗
Identification of two novel mutations in the 5'-untranslated region of H-ferritin using denaturing high performance liquid chromatography scanning.
BACKGROUND AND OBJECTIVES: Hereditary hyperferritinemia cataract syndrome is caused by mutations of the iron responsive elements (IREs) of L-ferritin mRNA. These alter the IRE structure and determine L-ferritin upregulation. IREs are located in 5'untranslated regions (5'UTR) of ferritin mRNAs. L-ferritin 5'UTR has been extensively studied and up to 21 different mutations have been identified. Only one mutation has been reported for H-ferritin 5'UTR; this mutation modified IRE structure and was apparently associated with high serum ferritin levels and iron overload. DESIGN AND METHODS: To identify other mutations in H ferritin 5'UTR we developed a fast DNA scanning method based on denaturing high performance liquid chromatography (HPLC). Five artificial DNA mutants were produced in order to validate the analytical conditions of the system for the identification of all mutations by single runs at 68 degrees C. The system was used to screen 660 DNA samples from subjects with high serum ferritin levels. RESULTS: Two abnormal patterns were identified carrying the mutations C20G and G34T. Structural data and the analysis of ferritin levels in red blood cells suggest that these mutations do not affect the functionality of the IRE. INTERPRETATION AND CONCLUSIONS: This large and first population analysis indicates that mutations in the H-ferritin 5'UTR are rare and do not seem to contribute to hyperferritinemia or iron overload.
5' Untranslated Regions↗
Genetic hyperferritinaemia and reticuloendothelial iron overload associated with a three base pair deletion in the coding region of the ferroportin gene (SLC11A3).
Iron overload may predominantly involve parenchymal or reticuloendothelial cells, the prototype of parenchymal iron overload being HFE-related genetic haemochromatosis. We studied a family with autosomal dominant hyperferritinaemia in whom the proband showed selective iron accumulation in the Kupffer cells on liver biopsy. Analysis of L and H ferritin genes excluded mutations responsible for hereditary hyperferritinaemia/cataract syndrome or similar translational disorders. Sequence analysis of the ferroportin gene (SLC11A3) in four individuals with hyperferritinaemia singled out a three base pair deletion in a region that contains four TTG repeats. This mutation removes a TTG unit from 780 to 791, and predicts the loss of one of three sequential valine residues 160-162. Denaturing high performance liquid chromatography can be used for its detection. SLC11A3 polymorphism analysis indicates that this probably represents a recurrent mutation due to slippage mispairing. Affected individuals may show marginally low serum iron and transferrin saturation, and young women may have marginally low haemoglobin concentration levels. Serum ferritin levels are directly related to age, but are 10-20 times higher than normal. Heterozygosity for the ferroportin Val 162 deletion represents the prototype of selective reticuloendothelial iron overload, and should be taken into account in the differential diagnosis of hereditary or congenital hyperferritinaemias.
Adult↗
Analysis of clinically relevant single-nucleotide polymorphisms by use of microelectronic array technology.
BACKGROUND: Microelectronic DNA chip devices represent an emerging technology for genotyping. We developed methods for detection of single-nucleotide polymorphisms (SNPs) in clinically relevant genes. METHODS: Primer pairs, with one containing a 5'-biotin group, were used to PCR-amplify the region encompassing the SNP to be interrogated. After denaturation, the biotinylated strand was electronically targeted to discrete sites on streptavidin-coated gel pads surfaces by use of a Nanogen Molecular Workstation. Allele-specific dye-labeled oligonucleotide reporters were used for detection of wild-type and variant sequences. Methods were developed for SNPs in genes, including factor VII, beta-globin, and the RET protooncogene. We genotyped 331 samples for five DNA variations in the factor VII gene, >600 samples from patients with beta-thalassemia, and 15 samples for mutations within the RET protooncogene. All samples were previously typed by various methods, including DNA sequence analysis, allele-specific PCR, and/or restriction enzyme digestion of PCR products. RESULTS: Analysis of amplified DNA required 4-6 h. After mismatched DNA was removed, signal-to-noise ratios were >5. More than 940 samples were typed with the microelectronic array platform, and results were totally concordant with results obtained previously by other genotyping methods. CONCLUSIONS: The described protocols detect SNPs of clinical interest with results comparable to those of other genotyping methods.
Factor VII↗