Barbara Foglieni

Mutational scanning of the ABCR gene with double-gradient denaturing-gradient gel electrophoresis (DG-DGGE) in Italian Stargardt disease patients

Mutations in the retina-specific ABC transporter (ABCR) gene are responsible for autosomal recessive Stargardt disease (arSTGD). Mutation detection efficiency in ABCR in arSTGD patients ranges between 30% and 66% in previously published studies, because of high allelic heterogeneity and technical limitations of the employed methods. Conditions were developed to screen the ABCR gene by double-gradient denaturing-gradient gel electrophoresis. The efficacy of this method was evaluated by analysis of DNA samples with previously characterized ABCR mutations. This approach was applied to mutation detection in 44 Italian arSTGD patients corresponding to 36 independent genomes, in order to assess the nature and frequency of the ABCR mutations in this ethnic group. In 34 of 36 (94.4%) STGD patients, 37 sequence changes were identified, including 26 missense, six frameshift, three splicing, and two nonsense variations. Among these, 20 had not been previously described. Several polymorphisms were detected in affected individuals and in matched controls. Our findings extend the spectrum of mutations identified in STGD patients and suggest the existence of a subset of molecular defects specific to the Italian population. The identification of at least two disease-associated mutations in four healthy control individuals indicates a higher than expected carrier frequency of variant ABCR alleles in the general population. Genotype-phenotype analysis in our series showed a possible correlation between the nature and location of some mutations and specific ophthalmoscopic features of STGD disease.

Case report: a subject with a mutation in the ATG start codon of L-ferritin has no haematological or neurological symptoms

Ferritin consists of two subunit types, H and L, which assemble in different proportions in a 24-mer protein.1 The H-subunit has ferroxidase activity and is mainly found in cell cytoplasm, where it has the major function of sequestering and detoxifying unwanted iron. The L-subunit has no catalytic activity on its own, but assists the functionality of the H-subunit2 and is also found in minor amounts in serum.3 Two types of genetic disorder are associated with mutations of the L-ferritin gene ( FTL ), both with autosomal dominant transmission. The first, hereditary hyperferritinaemia cataract syndrome (HHCS), is caused by mutations in the regulatory iron responsive element (IRE) in the 5′UTR of the transcript that reduce binding affinity to the iron regulatory proteins (IRPs) and lead to constitutive upregulation of the protein in tissue and serum.4–8 Subjects with the mutations show high levels of serum ferritin (500–2000 μg/l) and often early-onset bilateral cataracts6 likely caused by protein aggregation in the lens,9 but do not present alterations in iron metabolism. The disorder has been extensively studied and more than 21 different causative mutations have been identified.9,10 The second type of genetic disorder, neuroferritinopathy, is rare and few families have been identified. It is associated with an adenine insertion at position 460–461 in the coding region (exon 4) of the gene that causes a frame shift alteration of the C-terminus of the L-ferritin polypeptide.11 Affected subjects show late-onset movement disorders, iron deposition in the brain basal ganglia, and low serum ferritin levels.12,13 It is unclear whether the iron deposition in the brain is caused by a quantitative defect of L-ferritin or by an abnormal functionality caused by …

Integrated PCR amplification and detection processes on a Lab-on-Chip platform: a new advanced solution for molecular diagnostics.

Abstract Background: Several microdevices have been developed to perform only a single step of a genotyping process, such as PCR or detection by probe hybridization. Here, we describe a Lab-on-Chip (LoC) platform integrating a PCR amplification microreactor with a customable microarray for the detection of sequence variations on human genomic DNA. Methods: Preliminary work was focused on developing the single analytical steps including PCR and labeling strategies of the amplified product by conventional reference systems. The optimized protocols included a 1:4 forward:reverse primer ratio for asymmetric PCR, and Cy5-dCTP multiple incorporation for the generation of a labeled PCR product to be hybridized to complementary probes bound to the chip surface. Results: Final conditions were applied to the fully integrated LoC platform for the detection of the IVSI-110 G>A mutation in the human β-globin (HBB) gene associated with β-thalassemia, used as a model of genetic application, allowing for correct genotyping of 25 samples that were heterozygous, homozygous or wild-type for this mutation. Conclusions: The overall results show that the present platform is very promising for rapid identification of DNA sequence variations in an integrated, cost effective and convenient silicon chip format. Clin Chem Lab Med 2010;48:329–36.

Peptide-nucleic acid-mediated enriched polymerase chain reaction as a key point for non-invasive prenatal diagnosis of β-thalassemia

This study describes a novel approach to non-invasive pre-natal diagnosis of β-thalassemia based on microchip analysis of fetal DNA extracted from maternal plasma. The presence of fetal DNA in maternal plasma can be exploited to develop new procedures for non-invasive prenatal diagnosis. Tests to detect 7 frequent β-globin gene mutations in people of Mediterranean origin were applied to the analysis of maternal plasma in couples where parents carried different mutations. A mutant enrichment amplification protocol was optimized by using peptide nucleic acids (PNAs) to clamp maternal wild-type alleles. By this approach, 41 prenatal diagnoses were performed by microelectronic microchip analysis, with total concordance of results obtained on fetal DNA extracted from chorionic villi. Among these, 27/28 were also confirmed by direct sequencing and 4 by pyrosequencing.

Feasibility study for a microchip-based approach for noninvasive prenatal diagnosis of genetic diseases

Abstract: Fetal DNA in maternal plasma may represent a source of genetic material for prenatal noninvasive diagnosis of genetic diseases. We evaluated a cohort of physiological pregnancies to determine if fetal DNA can be retrieved at any gestational week in sufficient quantity to be analyzed with advanced mutation detection technologies. We performed fetal DNA quantification by real‐time polymerase chain reaction (PCR) on the SRY gene in 356 women sampled from 6 to 40 gestational weeks. Fetal DNA was retrieved at any week. All female fetuses were correctly identified. In 5 of 188 (2.6%) male‐bearing pregnancies, no amplification was obtained. For noninvasive testing, complete clearance of fetal DNA after delivery is mandatory. Long‐term persistence was not detected in women with previous sons or abortions. These findings confirm that maternal plasma may represent the optimal source of fetal genetic material. For noninvasive diagnosis of genetic diseases, we evaluated microchip technology. The detection limit for a minority allele determined by diluting a mutated DNA into a wild‐type plasma sample was 5 genome equivalents, indicating that the test might be applied to the identification of paternally inherited fetal alleles in maternal plasma. The addition of peptide nucleic acids (PNAs) to either the PCR reaction or the chip hybridization mixture allowed approximately 50% inhibition of wild‐type allele signals.

Scanning mutations of the 5'UTR regulatory sequence of L-ferritin by denaturing high-performance liquid chromatography: identification of new mutations.

Summary. Hereditary hyperferritinaemia cataract syndrome is an autosomal dominant disorder caused by heterogeneous mutations of the iron regulatory element (IRE) in the ferritin l‐chain mRNA. The mutations are rare and fast DNA scanning would facilitate diagnosis. The aim of the study was to compare the analytical performances of two fast DNA scanning techniques: denaturing high‐performance liquid chromatography (DHPLC) and double‐gradient denaturing gradient gel electrophoresis (DG‐DGGE). We analysed the sequence encoding the 5′ untranslated flanking region of ferritin l‐chain mRNA, which includes an IRE stem loop structure. The two systems unambiguously identified all the 12 accessible mutations in a single run, including the difficult C–G transversions. DHPLC and DG‐DGGE identified seven abnormal patterns in DNA samples from 47 subjects with unexplained hyperferritinaemia; all had mutations in the IRE sequence, including two not reported before: C36G and A37G. The scanning of 250 DNA samples from subjects genotyped for HFE led to the identification of four new mutations, all outside the IRE structure: C10T, C16T, C90T and del‐T156. We conclude that DHPLC, similar to DG‐DGGE, detects all the mutations in the l‐ferritin 5‘UTR sequence in a single run, and that various mutations occur outside the IRE structure.

A novel deletion of the l‐ferritin iron‐responsive element responsible for severe hereditary hyperferritinaemia–cataract syndrome

Summary. In the last few years, mutations that cause disease through increased efficiency of mRNA translation have been discovered. Hereditary hyperferritinaemia–cataract syndrome (HHCS) arises from various point mutations or deletions within the iron‐responsive element (IRE) in the 5′‐UTR of the l‐ferritin mRNA. Each unique mutation confers a characteristic degree of hyperferritinaemia and severity of cataract in affected individuals. We report a novel six‐nucleotide deletion identified in an Italian family presenting with elevated serum ferritin and early onset bilateral cataract. This deletion involves a sequence with a TCT repetition and may have occurred through a mechanism of slippage mispairing. Because of the above repetition, the observed mutation can be interpreted as deletion 22–27, 23–28, 24–29 or 25–30. Structural modelling predicted an IRE stem modification that is expected to markedly reduce the binding to iron‐regulatory proteins. A double‐gradient denaturing gradient gel electrophoresis (DG‐DGGE) method easily detected the above deletion.

Different approaches for noninvasive prenatal diagnosis of genetic diseases based on PNA-mediated enriched PCR

Abstract:  The aim of this work was to develop advanced and accessible protocols for noninvasive prenatal diagnosis of genetic diseases. We are evaluating different technologies for mutation detection, based on fluorescent probe hybridization of the amplified product and pyrosequencing, a technique that relies on the incorporation of nucleotides in a primer‐directed polymerase extension reaction. In a previous investigation, we have already proven that these approaches are sufficiently sensitive to detect a few copies of a minority‐mutated allele in the presence of an excess of wild‐type DNA, In this work, in order to further enhance the sensitivity, we have employed a mutant enrichment amplification strategy based on the use of peptide nucleic acids (PNAs). These DNA analogues bind wild‐type DNA, thus interfering with its amplification while still allowing the mutant DNA to become detectable. We have synthesized different PNAs, which are highly effective in clamping wild‐type DNA in the beta‐globin gene region, where four beta‐thalassemia mutations are located (IVSI.110, CD39, IVSI.1, IVSI.6) plus HbS. The fluorescence microchip readout allows us to monitor the extent of wild‐type allele inhibition, thus facilitating the assessment of the optimal PNA concentration.

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.

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.