Stefania Zampatti

Review of nutrient actions on age-related macular degeneration.

The actions of nutrients and related compounds on age-related macular degeneration (AMD) are explained in this review. The findings from 80 studies published since 2003 on the association between diet and supplements in AMD were reviewed. Antioxidants and other nutrients with an effect on AMD susceptibility include carotenoids (lutein and zeaxanthin, β-carotene), vitamins (vitamin A, E, C, D, B), mineral supplements (zinc, copper, selenium), dietary fatty acids [monounsaturated fatty acids, polyunsaturated fatty acids (PUFA both omega-3 PUFA and omega-6 PUFA), saturated fatty acids and cholesterol], and dietary carbohydrates. The literature revealed that many of these antioxidants and nutrients exert a protective role by functioning synergistically. Specifically, the use of dietary supplements with targeted actions can provide minimal benefits on the onset or progression of AMD; however, this does not appear to be particularly beneficial in healthy people. Furthermore, some supplements or nutrients have demonstrated discordant effects on AMD in some studies. Since intake of dietary supplements, as well as exposure to damaging environmental factors, is largely dependent on population habits (including dietary practices) and geographical localization, an overall healthy diet appears to be the best strategy in reducing the risk of developing AMD. As of now, the precise mechanism of action of certain nutrients in AMD prevention remains unclear. Thus, future studies are required to examine the effects that nutrients have on AMD and to determine which factors are most strongly correlated with reducing the risk of AMD or preventing its progression.

Typing of ARMS2 and CFH in age-related macular degeneration: case-control study and assessment of frequency in the Italian population.

OBJECTIVES To determine the effects of the polymorphisms CFH Tyr402His and ARMS2 del443ins54 on susceptibility to age-related macular degeneration (AMD) and to find the frequencies of these single-nucleotide polymorphisms in an Italian population that was not examined clinically. METHODS A total of 286 control subjects (126 men and 160 women) and 159 white patients (73 men and 86 women) harboring exudative AMD in 1 eye were recruited. A third group of 182 DNA samples from blood donors of the same geographical areas were also typed to assess the frequency of CFH Tyr402His and ARMS2 del443ins54 polymorphisms in the general population. The data were analyzed statistically by a standard 2 x 2 table, Fisher exact tests, and odds ratios. RESULTS The deletion-insertion at chromosome 10q26 (del443ins54) showed the strongest association with AMD in terms of both P value and odds ratio (P = 2.7 x 10(-15); odds ratio = 3.25), and a highly significant association was also confirmed for Tyr402His at the CFH locus (P = 9.9 x 10(-13); odds ratio = 2.86). We found no differences in allele and genotype association between classic and occult choroidal neovascularization. We also observed that 39% of the samples in the general Italian population were at least 5.4 times more likely than control subjects to develop AMD. CONCLUSIONS To our knowledge, this is the first confirmation of the association of del443ins54 in Italian patients with AMD, and we also confirmed the association of Tyr402His with CFH. Genetic analysis of the general population suggested that analysis of the ARMS2 and CFH risk alleles alone may be helpful in differentiating high-risk individuals (odds ratio > 5.00) from low-risk individuals (odds ratio < 0.45). CLINICAL RELEVANCE Individuals at high risk for developing AMD could be identified and selected for specific prevention programs. In this context, the development of prevention programs based on dietary antioxidants or on close monitoring of at-risk individuals should be considered or suggested.

Haplotypes in IL-8 Gene Are Associated to Age-Related Macular Degeneration: A Case-Control Study

Background Age-related macular degeneration (AMD) is the main cause of blindness in the developed world. The etiology of AMD is multifactorial due to the interaction between genetic and environmental factors. IL-8 has a role in inflammation and angiogenesis; we report the genetic characterization of IL-8 allele architecture and evaluate the role of SNPs or haplotypes in the susceptibility to wet AMD, case-control study. Methods Case-control study including 721 AMD patients and 660 controls becoming from Italian population. Genotyping was carried out by Real Time-PCR. Differences in the frequencies were estimated by the chi-square test. Direct sequencing was carried out by capillary electrophoresis trough ABI3130xl. Results rs2227306 showed a p–value of 4.15*10−5 and an Odds Ratio (OR) for T allele of 1.39 [1.19–1.62]. After these positive results, we sequenced the entire IL-8 regulatory and coding regions of 60 patients and 30 controls stratified for their genotype at rs2227306. We defined two different haplotypes involving rs4073 (A/T), rs2227306 (C/T), rs2227346 (C/T) and rs1126647 (A/T): A-T-T-T (p-value: 2.08*10−9; OR: 1.68 [1.43–1.97]) and T-C-C-A (p-value: 7.07*10−11; OR: 0.60 [0.51–0.70]). To further investigate a potential functional role of associated haplotypes, we performed an expression study on RNA extracted from whole blood of 75 donors to verify a possible direct correlation between haplotype and gene expression, failing to reveal significant differences. Conclusions These results suggest a possible secondary role of IL-8 gene in the development of the disease. This paper outlines the importance of association between inflammation and AMD. Moreover IL-8 is a new susceptibility genomic biomarker of AMD.

De Barsy Syndrome: a genetically heterogeneous autosomal recessive cutis laxa syndrome related to P5CS and PYCR1 dysfunction.

De Barsy Syndrome: A Genetically Heterogeneous Autosomal Recessive Cutis Laxa SyndromeRelated to P5CS and PYCR1 Dysfunction Stefania Zampatti, Marco Castori, Bjoern Fischer, Paola Ferrari, Livia Garavelli, Carlo Dionisi-Vici, Emanuele Agolini, Anita Wischmeijer, Eva Morava, Giuseppe Novelli, Johannes H€aberle, Uwe Kornak,** and Francesco Brancati* Department of Biopathology and Diagnostic Imaging, Tor Vergata University, Rome, Italy Department of Molecular Medicine, Medical Genetics, San Camillo-Forlanini Hospital, Sapienza University, Rome, Italy Institute for Medical and Human Genetics, Charit e-Universitaetsmedizin, Berlin, Germany Department of Pediatrics, University of Modena and Reggio Emilia, Modena, Italy Clinical Genetics Unit, Santa Maria Nuova Hospital IRCCS, Reggio Emilia, Italy Division of Metabolism, Bambino Gesù Pediatric Hospital, Rome, Italy IRCCS Casa Sollievo della Sofferenza, Istituto Mendel, Rome, Italy Department of Molecular Medicine, Sapienza University, Rome, Italy Genetic Unit, Sant’Orsola Malpighi Hospital, Bologna, Italy Department of Pediatrics, UMC Radboud Nijmegen, Nijmegen, The Netherlands Italian National Agency for Evaluation of University and Research (ANVUR), Rome, Italy Division of Metabolism, Kinderspital Zurich, Pediatric Research Centre, Zurich, Switzerland Max Planck Institute for Molecular Genetics, Berlin, Germany

Whole genome amplification and real-time PCR in forensic casework

BackgroundWGA (Whole Genome Amplification) in forensic genetics can eliminate the technical limitations arising from low amounts of genomic DNA (gDNA). However, it has not been used to date because any amplification bias generated may complicate the interpretation of results. Our aim in this paper was to assess the applicability of MDA to forensic SNP genotyping by performing a comparative analysis of genomic and amplified DNA samples. A 26-SNPs TaqMan panel specifically designed for low copy number (LCN) and/or severely degraded genomic DNA was typed on 100 genomic as well as amplified DNA samples.ResultsAliquots containing 1, 0.1 and 0.01 ng each of 100 DNA samples were typed for a 26-SNPs panel. Similar aliquots of the same DNA samples underwent multiple displacement amplification (MDA) before being typed for the same panel. Genomic DNA samples showed 0% PCR failure rate for all three dilutions, whilst the PCR failure rate of the amplified DNA samples was 0% for the 1 ng and 0.1 ng dilutions and 0.077% for the 0.01 ng dilution. The genotyping results of both the amplified and genomic DNA samples were also compared with reference genotypes of the same samples obtained by direct sequencing. The genomic DNA samples showed genotype concordance rates of 100% for all three dilutions while the concordance rates of the amplified DNA samples were 100% for the 1 ng and 0.1 ng dilutions and 99.923% for the 0.01 ng dilution. Moreover, ten artificially-degraded DNA samples, which gave no results when analyzed by current forensic methods, were also amplified by MDA and genotyped with 100% concordance.ConclusionWe investigated the suitability of MDA material for forensic SNP typing. Comparative analysis of amplified and genomic DNA samples showed that a large number of SNPs could be accurately typed starting from just 0.01 ng of template. We found that the MDA genotyping call and accuracy rates were only slightly lower than those for genomic DNA. Indeed, when 10 pg of input DNA was used in MDA, we obtained 99.923% concordance, indicating a genotyping error rate of 1/1299 (7.7 × 10-4). This is quite similar to the genotyping error rate of STRs used in current forensic analysis. Such efficiency and accuracy of SNP typing of amplified DNA suggest that MDA can also generate large amounts of genome-equivalent DNA from a minimal amount of input DNA. These results show for the first time that MDA material is suitable for SNP-based forensic protocols and in general when samples fail to give interpretable STR results.

The pharmacogenomic HLA biomarker associated to adverse abacavir reactions: Comparative analysis of different genotyping methods

Many pharmacogenomic biomarkers (PGBM) were identified and translated into clinical practice, affecting the usage of drugs via label updates. In this context, abacavir is one of the most brilliant examples of pharmacogenetic studies translated into clinical practice. Pharmacogenetic studies have revealed that abacavir HSRs are highly associated with the major histocompatibility complex class I. Large studies established the effectiveness of prospective HLA-B*57:01 screening to prevent HSRs to abacavir. Accordingly to these results the abacavir label has been modified: the European Medicines Agency (EMA) and the FDA recommend/suggested that the administration of abacavir must be preceded by a specific genotyping test. The HLA locus is extremely polymorphic, exhibiting many closely related alleles, making it difficult to discriminate HLA-B*57:01 from other related alleles, and a number of different molecular techniques have been developed recently to detect the presence of HLA-B*57:01. In this review, we provide a summary of the available techniques used by laboratories to genotype HLA-B*57:01, outlining the scientific and pharmacoeconomics pros and cons.

Four Copies of SNCA Responsible for Autosomal Dominant Parkinson's Disease in Two Italian Siblings.

Background. Parkinsons disease (PD) is mostly characterized by alpha-synuclein (SNCA) aggregation and loss of nigrostriatal dopamine-containing neurons. In this study a novel SNCA multiplication is described in two siblings affected by severe parkinsonism featuring early onset dyskinesia, psychiatric symptoms, and cognitive deterioration. Methods. SNCA dosage was performed using High-Density Comparative Genomic Hybridization Array (CGH-Array), Multiple Ligation Dependent Probe Amplification (MLPA), and Quantitative PCR (qPCR). Genetic analysis was associated with clinical evaluation. Results. Genetic analysis of siblings showed for the first time a 351 Kb triplication containing SNCA gene along with 6 exons of MMRN1 gene in 4q22.1 and a duplication of 1,29 Mb of a genomic region flanking the triplication. Conclusions. The identification of this family indicates a novel mechanism of SNCA gene multiplication, which confirms the genomic instability in this region and provides data on the genotype-phenotype correlation in PD patients.

Direct PCR: a new pharmacogenetic approach for the inexpensive testing of HLA-B*57:01.

One of the most successful applications of pharmacogenetics research is the genetic screening for HLA-B*57:01, strongly associated with an increased risk to develop hypersensitivity reaction in HIV-positive patients following abacavir administration. Taking into consideration the limits of current genotyping methodologies, we have developed and validated (150 buccal swabs) an inexpensive pharmacogenetic approach for HLA-B*57:01 typing. In our assay DNA extraction and amplification are combined in one single step (direct PCR protocol), which is performed directly on the biological sample without the need of extraction and sequencing passages. The amplicons obtained by direct PCR can be easily separated on the agarose gel under ultraviolet. As per our results, the direct PCR represents a good alternative to the traditional methods of HLA-B*57:01 pharmacogenetic test, especially for those laboratories or countries where currently available approaches are often not available or not affordable. Furthermore it is an innovative approach, promoting a personalized, safer and cost-effective therapy.

A fluorescence-based sequence-specific primer PCR for the screening of HLA-B*57:01

Abacavir (ABC) is an antiretroviral drug highly effective in the treatment of HIV, but its intake can cause severe hypersensitivity reaction (HSR). A strong association between HLA‐B*57:01 and ABC HSRs was reported by several studies, which demonstrated that HLA‐B*57:01 screening had a 100% negative predictive value and that it could accurately identify patients at high risk of ABC HSRs. We propose a new sequence‐specific primer PCR assay based on fluorescence detection through CE which is highly sensitive, allowing the use of non‐infective sources of DNA such as saliva and buccal swabs, in addition to blood and reproducible, allowing automation of the analytical process. The results of our study were first compared with a standard sequence‐specific primer PCR technique and reported a concordance of 100%, and then a blind external validation further confirmed the accuracy of our method.

The Genetics and the Genomics of Primary Congenital Glaucoma

The sight is one of the five senses allowing an autonomous and high-quality life, so that alterations of any ocular component may result in several clinical phenotypes (from conjunctivitis to severe vision loss and irreversible blindness). Most parts of clinical phenotypes have been significantly associated with mutations in genes regulating the normal formation and maturation of the anterior segments of the eye. Among the eye anterior segment disorders, special attention is given to Glaucoma as it represents one of the major causes of bilateral blindness in the world, with an onset due to Mendelian or multifactorial genetic-causative traits. This review will point out the attention on the Primary Congenital Glaucoma (PCG), which is usually transmitted according to an autosomal-recessive inheritance pattern. Taking into consideration the genetic component of the PCG, it is possible to observe a strong heterogeneity concerning the disease-associated loci (GLC3), penetrance defects, and expressivity of the disease. Given the strong PGC heterogeneity, pre- and posttest genetic counseling plays an essential role in the achievement of an appropriate management of PCG, in terms of medical, social, and psychological impact of the disease.