Claudia Strafella

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.

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.

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.

Two molecular assays for the rapid and inexpensive detection of GJB2 and GJB6 mutations.

The hypoacusia can be classified in two clinical forms: Syndromic (SHL) and Nonsyndromic (NSHL). In particular, the NSHL describes the 70–80% of hypoacusia cases and it is mainly due to genetic factors, which are causative of the deafness at the birth. The genetic hypoacusia presents different inheritance patterns: autosomal dominant (20%), autosomal recessive (80%), X‐linked (1%), and mitochondrial (1%), respectively. To date, about 35 deafness‐causative genes have been identified and most of them codify for connexin transmembrane proteins. Approximately 1:2500 children with NSHL carries mutations in the GJB2 and GJB6 (13q12) genes, which code for connexin 26 (Cx26) and connexin 30 (Cx30), respectively. In the Caucasian population, the most common mutations are 35delG, M34T and 167delT, and D13S1830. Given the frequency distribution of the four mutations in the Caucasian population and the pathogenic connection with NSHL, the development of accurate, rapid, and “low‐cost” molecular assays should be strongly encouraged. To this purpose, we set up two different molecular assays (namely the Cx26 and Cx26‐30 molecular assays) for the fast and inexpensive detection of 35delG, M34T, 167delT, and D13S1830 mutations. Both the molecular approaches showed to be accurate, sensitive, reproducible, and “low‐cost” alternatives for the proper evaluation of the GJB2 and GJB6 genes, which are causative of NSHL. In conclusion, the Cx26 and Cx26‐30 molecular assays can be applied to individual, preconception, prenatal, or postnatal screening for the causative‐mutations of NSHL.

KIF3A and IL-4 are disease-specific biomarkers for psoriatic arthritis susceptibility

To date, the genes associated with Psoriatic Arthritis (PsA) are principally involved in inflammation, immune response and epidermal differentiation, without any information about the relationship between disease and bone metabolism genes. Our work was focused on 5q31 locus, which contains several genetic variants significantly associated with PsA. The study involved 1526 subjects (500 PsA, 426 PsV, 600 controls). The region was evaluated by selecting and genotyping the SNPs of interest by Real Time PCR and direct sequencing. The results were subjected to biostatistic and bioinformatic analysis. The case-control study highlighted a significant association between KIF3A/IL-4 and PsA, but not with PsV (Psoriasis Vulgaris) patients. In addition, the haplotype analysis revealed two haplotypes significantly associated with PsA susceptibility. The Linkage Disequilibrium (LD) study showed the presence of a specific block in high LD within 132,692,628-132,737,638 bp of 5q31, giving additional evidence of specific association of the 5q31 region in PsA patients. Moreover, KIF3A expression was assessed by immunohistochemistry assays which showed a marked and significant difference of KIF3A expression between pathological and normal tissues. Our analysis described KIF3A and IL-4 as novel susceptibility genes for PsA, suggesting a clear implication of bone metabolism genes in the disease etiopathogenesis.

Uncovering genetic and non-genetic biomarkers specific for exudative age-related macular degeneration: significant association of twelve variants

Age-related Macular Degeneration (AMD) represents one of the most sight-threatening diseases in developed countries that substantially impacts the patients’ lifestyle by compromising everyday activities, such as reading and driving. In this context, understanding the prevalence, burden, and population-specific risk/protective factors of AMD is essential for adequate health care planning and provision. Our work aimed to characterize exudative AMD in Italian population and to identify the susceptibility/protective factors (genetic variants, age, sex, smoking and dietary habits) which are specific for the onset of disease. Our study involved a cohort of 1976 subjects, including 976 patients affected with exudative AMD and 1000 control subjects. In particular, the sample cohort has been subjected to a large genotyping analysis of 20 genetic variants which are known to be associated with AMD among European and Asiatic populations. This analysis revealed that 8 genetic variants (CFH, ARMS2, IL-8, TIMP3, SLC16A8, RAD51B, VEGFA and COL8A1) were significantly associated with AMD susceptibility. Successively, we performed a multivariate analysis, considering both genetic and non-genetic data available for our sample cohort. The multivariate analysis showed that age, smoking, dietary habits and sex, together with the genetic variants, were significantly associated with AMD in our population. Altogether, these data represent a starting point for the set-up of adequate preventive and personalized strategies aimed to decrease the burden of disease and improve the patients’ quality of life.

Application of Precision Medicine in Neurodegenerative Diseases

One of the main challenges for healthcare systems is the increasing prevalence of neurodegenerative pathologies together with the rapidly aging populations. The enormous progresses made in the field of biomedical research and informatics have been crucial for improving the knowledge of how genes, epigenetic modifications, aging, nutrition, drugs and microbiome impact health and disease. In fact, the availability of high technology and computational facilities for large-scale analysis enabled a deeper investigation of neurodegenerative disorders, providing a more comprehensive overview of disease and encouraging the development of a precision medicine approach for these pathologies. On this subject, the creation of collaborative networks among medical centers, research institutes and highly qualified specialists can be decisive for moving the precision medicine from the bench to the bedside. To this purpose, the present review has been thought to discuss the main components which may be part of precise and personalized treatment programs applied to neurodegenerative disorders. Parkinson Disease will be taken as an example to understand how precision medicine approach can be clinically useful and provide substantial benefit to patients. In this perspective, the realization of web-based networks can be decisive for the implementation of precision medicine strategies across different specialized centers as well as for supporting clinical/therapeutical decisions and promoting a more preventive and participative medicine for neurodegenerative disorders. These collaborative networks are essentially addressed to find innovative, sustainable and effective strategies able to provide optimal and safer therapies, discriminate at risk individuals, identify patients at preclinical or early stage of disease, set-up individualized and preventative strategies for improving prognosis and patients quality of life.

Pharmacogenomics of multifactorial diseases: a focus on psoriatic arthritis.

This review will outline the current pharmacogenomics knowledge about psoriatic arthritis with a special attention to the perspectives and the challenges for its implementation in the clinical practice. To date, different drugs have been developed to contrast the symptoms and the progression of psoriatic arthritis. However, patients have shown high variability of drug response in relation to their genetic makeup. In this context, the advances made in the knowledge and the potentialities of genome-drugs associations paved the path for the development of a precision medicine. In fact, these associations may be successfully combined with the environment information to provide new strategies able to prevent and improve the disease management as well as to enhance the patients quality of life.