Raffaella Cascella

Full Sequencing of the FLG Gene in Italian Patients with Atopic Eczema: Evidence of New Mutations, but Lack of an Association

TO THE EDITOR Atopic eczema (AE) (OMIM %603165) is the most common chronic inflammatory skin disease, characterized by xerosis, pruritus, and erythematous lesions with increased transepidermal water loss. In recent years, it has been suggested that the epidermal skin barrier has a significant role in AE disease susceptibility and severity (Smith et al., 2006; Cork et al., 2009). Sandilands et al. (2006) demonstrated that null mutations within the filaggrin gene (FLG) strongly predispose individuals to AE. Two FLG null alleles (R501X and 2282del4) have been shown to be significantly associated with AE in several European populations (Palmer et al., 2006; Weidinger et al., 2007). Recently, a meta-analysis of the most common FLG variants in European populations, involving 5,791 eczema cases and 26,454 controls (Rodriguez et al., 2009), revealed that there is a high risk conferred by R501X and 2282del4 across the studies, with an overall odds ratio of 3.14 and 2.78, respectively. Indeed, large differences in carrier frequencies exist across Europe, ranging from 1.4% in an Italian population (Giardina et al., 2008) to 63% in an Irish population (Palmer et al., 2006). Recently, we observed that in Italian patients the frequencies of R501X and 2282del4 are strongly reduced with respect to those described in other patients of European origin, and the frequencies are similar between cases and controls (0.6 vs. 0.0% and 0.9 vs. 0.5%, respectively). In order to determine whether other mutations located elsewhere in FLG confer risk to AE, we performed a full sequencing of FLG in Italian patients. We performed a sequencing of the full FLG gene in a cohort of 220 Italian AE patients (recruited by IDI-Istituto Dermopatico dell’Immacolata and Fatebenefratelli Hospital). We then determined the frequency of variations and mutations in a cohort of 201 healthy subjects. The diagnosis of AE in our case cohort was made by experienced dermatologists or by a pediatric allergologist. The cohort consisted of 85% of cases with the intrinsic subtype and 15% with the extrinsic form of AE. These subtypes and severity of AE have been established based on the total IgE level (extrinsic subtype 4500 ng l ) and using the scoring atopic dermatitis. Further clinical details of Italian patients are available in Abbreviations: AE, atopic eczema; FLG, filaggrin gene; LD, linkage disequilibrium

Age-Related Macular Degeneration: Insights into Inflammatory Genes

Age-related macular degeneration (AMD) is a progressive neurodegenerative disease that affects approximately 8.7% of elderly people worldwide (>55 years old). AMD is characterized by a multifactorial aetiology that involves several genetic and environmental risk factors (genes, ageing, smoking, family history, dietary habits, oxidative stress, and hypertension). In particular, ageing and cigarette smoking (including oxidative compounds and reactive oxygen species) have been shown to significantly increase susceptibility to the disease. Furthermore, different genes (CFH, CFI, C2, C3, IL-6, IL-8, and ARMS2) that play a crucial role in the inflammatory pathway have been associated with AMD risk. Several genetic and molecular studies have indicated the participation of inflammatory molecules (cytokines and chemokines), immune cells (macrophages), and complement proteins in the development and progression of the disease. Taking into consideration the genetic and molecular background, this review highlights the genetic role of inflammatory genes involved in AMD pathogenesis and progression.

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.

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.

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.

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.

Association of KIF3A, but not OVOL1 and ACTL9, with atopic eczema in Italian patients

Background  Atopic eczema (AE) (OMIM %603165) is the most common chronic inflammatory skin disease characterized by xerosis, pruritus, and erythematous lesions with increased transepidermal water loss. It’s a complex disease due to the interaction between environmental and genetics factors. To date, different loci have been related to the disease.

A multiplex molecular assay for the detection of uniparental disomy for human chromosome 7.

Uniparental disomy (UPD) describes the inheritance of both homologues of a pair of chromosomes from only one parent. During the last two decades, the clinical impact of UPD and associated imprinting disorders, such as Prader‐Willi syndrome (PWS) and Angelman syndrome (AS) increasingly have come to our attention. About 25% of PWS and 3%–5% of AS are a consequence of UPD with the resulting phenotype generated from the parent of origin of the disomic pair of chromosomes 15. Chromosome 15 UPD testing is relevant in various prenatal diagnostic conditions including apparent confined placental mosaicism, homologous and nonhomologous Robertsonian translocations involving chromosome 15 and 14, and as genomic biomarker for detecting chromosome origin. In this work we developed and validated a two fluorescent STRs multiplex assay for a rapid, economic and fully informative detection of UPD 15 by capillary electrophoresis.

Comparative analysis between saliva and buccal swabs as source of DNA: lesson from HLA-B*57:01 testing

AIM Our work aimed to designate the optimal DNA source for pharmacogenetic assays, such as the screening for HLA-B*57:01 allele. MATERIALS & METHODS A saliva and four buccal swab samples were taken from 104 patients. All the samples were stored at different time and temperature conditions and then genotyped for the HLA-B*57:01 allele by SSP-PCR and classical/capillary electrophoresis. RESULTS The genotyping analysis reported different performance rates depending on the storage conditions of the samples. Given our results, the buccal swab demonstrated to be more resistant and stable in time with respect to the saliva. CONCLUSION Our investigation designates the buccal swab as the optimal DNA source for pharmacogenetic assays in terms of resistance, low infectivity, low-invasiveness and easy sampling, and safe transport in centralized medical centers providing specialized pharmacogenetic tests.

Assessing individual risk for AMD with genetic counseling, family history, and genetic testing

PurposeThe goal was to develop a simple model for predicting the individual risk profile for age-related macular degeneration (AMD) on the basis of genetic information, disease family history, and smoking habits.Patients and methodsThe study enrolled 151 AMD patients following specific clinical and environmental inclusion criteria: age >55 years, positive family history for AMD, presence of at least one first-degree relative affected by AMD, and smoking habits. All of the samples were genotyped for rs1061170 (CFH) and rs10490924 (ARMS2) with a TaqMan assay, using a 7500 Fast Real Time PCR device. Statistical analysis was subsequently employed to calculate the real individual risk (OR) based on the genetic data (ORgn), family history (ORf), and smoking habits (ORsm).Results and conclusionThe combination of ORgn, ORf, and ORsm allowed the calculation of the Ort that represented the realistic individual risk for developing AMD. In this report, we present a computational model for the estimation of the individual risk for AMD. Moreover, we show that the average distribution of risk alleles in the general population and the knowledge of parents’ genotype can be decisive to assess the real disease risk. In this contest, genetic counseling is crucial to provide the patients with an understanding of their individual risk and the availability for preventive actions.