Biomedical subjects

Stefania Stenirri

Publications and source records attributed to Stefania Stenirri.

6 recordsLinked to original sources

PubMed2006

De novo deletion removes a conserved motif in the C-terminus of ABCA4 and results in cone-rod dystrophy.

BACKGROUND: Mutations in the retina-specific ABC transporter (ABCA4) gene are associated with different types of macular degeneration, including Stargardt disease, cone-rod dystrophy, Fundus flavimaculatus, Retinitis pigmentosa and probably age-related macular degeneration. METHODS: Screening for mutations in the ABCA4 gene was performed using denaturing high-performance liquid chromatography and direct sequencing. RESULTS: We describe the identification of a new de novo 44-bp deletion in an Italian patient affected by cone-rod dystrophy. The mutation, located in intron 48 of the ABCA4 gene, is predicted to cause exon 49 skipping, resulting in loss of the C-terminus of the ABCA4 protein. Interestingly, exon 49 also codes for a highly conserved VFVNFA motif, which has been demonstrated to be essential for the activity of ABCA1, another gene of the ABC transporter family. The presence of CT repeats at the breakpoints might have facilitated the generation of the deletion through a slippage mispairing mechanism. CONCLUSIONS: The new 6730-16del44 deletion is the first de novo mutation associated with cone-rod dystrophy and may contribute to a better understanding of the role of ABCA4 mutations in macular dystrophies.

ATP-Binding Cassette Transporters↗

PubMed2005

Single-nucleotide polymorphism and mutation identification by the nanogen microelectronic chip technology.

The present chapter describes a microarray technology developed by Nanogen Inc., for the identification of DNA variations based on the use of microelectronics. The NMW 1000 NanoChip Molecular Biology Workstation allows the active deposition and concentration of charged biotinylated molecules on designated test sites. The DNA at each pad is then hybridized with specific oligonucleotide probes, complementary to normal or mutant sequences, that labeled with Cy3 or Cy5 dyes, respectively. The array is imaged, and fluorescence signals are scanned, monitored, and quantified by highly developed, digital image-processing procedures. The experimental steps to be performed for the development and execution of a microchip assay are described. Attention is focused on the fundamental aspects of probe design, and guidelines and useful suggestions are given. Protocols for sample preparation, addressing, reporting, and data analysis are also detailed.

Base Sequence↗

PubMed2005

Molecular diagnostics by microelectronic microchips.

Molecular diagnostics is being revolutionized by the development of highly advanced technologies for DNA and RNA testing. One of the most important challenges is the integration of microelectronics to microchip-based nucleic acid technologies. The specific characteristics of these microsystems make the miniaturization and automation of any step of a molecular diagnostic procedure possible. This review describes the application of microelectronics to all the processes involved in a genetic test, particularly to sample preparation, DNA amplification and sequence variation detection.

Animals↗

PubMed2004

Denaturing HPLC profiling of the ABCA4 gene for reliable detection of allelic variations.

BACKGROUND: Mutations in the retina-specific ABC transporter (ABCA4) gene have been associated with several forms of macular degenerations. Because the high complexity of the molecular genotype makes scanning of the ABCA4 gene cumbersome, we describe here the first use of denaturing HPLC (DHPLC) to screen for ABCA4 mutations. METHODS: Temperature conditions were designed for all 50 exons based on effective separation of 83 samples carrying 86 sequence variations and 19 mutagenized controls. For validation, samples from 23 previously characterized Stargardt patients were subjected to DHPLC profiling. Subsequently, samples from a cohort of 30 patients affected by various forms of macular degeneration were subjected to DHPLC scanning under the same conditions. RESULTS: DHPLC profiling not only identified all 132 sequence alterations previously detected by double-gradient denaturing gradient gel electrophoresis but also identified 5 sequence alterations that this approach had missed. Moreover, DHPLC scanning of an additional panel of 30 previously untested patients led to the identification of 26 different mutations and 29 polymorphisms, accounting for 203 sequence variations on 29 of the 30 patients screened. In total, the DHPLC approach allowed us to identify 16 mutations that had never been reported before. CONCLUSIONS: These results provide strong support for the use of DHPLC for molecular characterization of the ABCA4 gene.

ATP-Binding Cassette Transporters↗

PubMed2003

Denaturing HPLC analysis of DNA deletions and insertions.

Denaturing HPLC (DHPLC) is a useful technique for the fast screening of known and unknown heterozygous gene mutations. Most DNA mutations causing genetic disorders consist of nucleotide substitutions, but insertions and deletions occur, albeit less frequently. The heteroduplexes with insertions/deletions have gaps that may affect molecular stability differently from the mismatches caused by substitutions. Therefore, gaps and mismatches may be distinguished by DHPLC analysis, which is based on the differential thermal stability of amplicons with different characteristics. To verify this hypothesis, we examined 12 DNA samples containing insertions and deletions of different sizes (one to 29 residues) from four different genes (ABCA4, CFTR, FTL, and SLC11A3). We found that all of them were detected by DHPLC runs at 50 degrees C, which is considered a non-denaturing temperature, as well as by runs at the temperature optimized for mismatch recognition. The finding confirms that gaps reduce heteroduplex stability more than mismatches, and indicates that DHPLC analysis at low temperature may be applied to distinguish DNA deletions/insertions from substitutions.

5' Untranslated Regions↗

PubMed2003

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↗