Bioinformatics e Biostatistics applied to research in pediatric genetic disease. Clinical evidence in IFNλ4 polymorphisms associated with HCV infection in patients with beta thalassemia and WGCNA analysis weighted for IFNλ4 genotype rs12979860 to detect RPL9P18 as hub in HCV infected cell.

Genome-wide association studies have identified host genetic variation to be critical for spontaneous clearance and treatment response in patients infected with hepatitis C virus (HCV). We demonstrated the same in patients with thalassemia major infected by genotype 1b of HCV. In the present first part study we retrospectively analyzed 368 anti-HCV positive patients with beta-thalassemia in two Italian major thalassemic centers (Cagliari and Turin). The strongest IFNλ4 SNP found associated with HCV was rs12979860 where C/C genotype was related to response to the interferon treatment and, above all, to spontaneous clearance of the virus. However, the positive predictive power was stronger for viral persistence than spontaneous clearance indeed TT allele was more predictive than CC. Another polymorphism rs4803221 was analyzed because had independent effects respect to rs12979860. The haplotype tagged by SNP rs12979860 and rs4803221 significantly could improve the viral clearance prediction in infected patients. Neither necrotic-inflammation or bilirubin values in the chronic phase of the hepatitis C were related to IFNλ4 polymorphisms. No relation among IFNλ4 polymorphisms and fibrosis stage directly shown by the liver biopsy was found. Second part of our study was to identify hub genes associated in pathways closely related to IFNλ4 variants in HCV response. We used gene expression profile data of GSE54648, downloaded from Gene Expression Omnibus (GEO). We focused our attention on expression genes differential between rs12979860 unfavorable TT genotype and favorable CC genotype, using weighted gene expression network analysis (WGCNA - R package). Significant modules were selected using the clustering analysis. At the final the best significant module was “black” module. Its pathways were involved in translation mechanisms such as translation termination, translation elongation, nuclear-transcribed mRNA catabolic process, cellular protein complex disassembly, therefore biological mechanisms that occur inside ribosome. We discovered RPL9P18 pseudogene as a hub potentially related in inhibition of spontaneous clearance and furthermore likely involved in drug treatment inhibition. Our result suggests an active role for ribosome pseudogene in innate antiviral response probably during ISG (IFN-stimulated genes) translation. Moreover, through co-expression analysis we demonstrate a new possible role of IFNλ4 genotype in HCV infection, associate with expression of ribosomal pathways

Forage mixture productivity and silage quality from a grass/legume intercrop in a semiarid Mediterranean environment

In semiarid environments of the Mediterranean region, intercropping is a sustainable agricultural system of long standing. In this area, the pea (Pisum sativum L.) is one of the most commonly grown legume crops. Little information is available on the quality of silages to be obtained from forage mixtures of pea intercropped with cereals or annual grasses. In this study, two experiments were conducted over the course of two growing seasons in Sicily (Italy) with the aim to determine the biomass production of forage crop mixtures and assess, only in the second experiment, the silage quality of grass and legumes. Four cereals and one annual grass species were grown in pure stand and in mixture with pea, and their main agronomic traits were determined. The land equivalent ratio (LER), competitive ratio, and aggressivity index were also calculated. A number of parameters were considered to assess the quality of silage obtained from fermented biomasses derived from pea–ryegrass (Lolium multiflorum Lam. var. Westerwoldicum) intercropping. In the first experiment, the best performance between the intercrops was recorded for the pea–wheat mixture. The total LER calculated for fodder yields was always greater than 1, indicating crop yield advantages ranging from 2.0% to 47.0%. In the second experiment, the pea–ryegrass mixture appeared to respond well, depending on plant arrangement and seeding ratio factors: the ratios 50:50 and 100:50 showed the greatest crop yield advantages, of 12.0% and 11.0%, respectively. All silages revealed a very good suitability of a pea–ryegrass intercropping system with high-quality silage production in the Southern Mediterranean region

Quality assurance of genetic laboratories and the EBTNA practice certification, a simple standardization assurance system for a laboratory network

Abstract Analytical laboratory results greatly influence medical diagnosis, about 70% of medical decisions are based on laboratory results. Quality assurance and quality control are designed to detect and correct errors in a laboratory's analytical process to ensure both the reliability and accuracy of test results. Unreliable performance can result in misdiagnosis and delayed treatment. Furthermore, improved quality guarantees increased productivity at a lower cost. Quality assurance programmes include internal quality control, external quality assessment, proficiency surveillance and standardization. It is necessary to try to ensure compliance with the requirements of the standards at all levels of the process. The sources of these standards are the International Standards Organization (ISO), national standards bodies, guidelines from professional organisations, accreditation bodies and governmental regulations. Laboratory networks increase the performance of laboratories in support of diagnostic screening programme. It is essential that genetic laboratories of a network have procedures underpinned by a robust quality assurance system to minimize errors and to reassure the clinicians and the patients that international standards are being met. This article provides an overview of the bases of quality assurance and its importance in genetic tests and it reports the EBTNA quality assurance system which is a clear and simple system available for access to adequate standardization of a genetic laboratory's network

Optimization of long-range PCR protocol to prepare filaggrin exon 3 libraries for PacBio long-read sequencing

BackgroundThe filaggrin (FLG) protein, encoded by the FLG gene, is an intermediate filament-associated protein that plays a crucial role in the terminal stages of human epidermal differentiation. Loss-of-function mutations in the FLG exon 3 have been associated with skin diseases. The identification of causative mutations is challenging, due to the high sequence homology within its exon 3 (12,753 bp), which includes 10 to 12 filaggrin tandem repeats. With this study we aimed to obtain the whole FLG exon 3 sequence through PacBio technology, once 13-kb amplicons have been generated.Methods and resultsFor the preparation of SMRTbell libraries to be sequenced using PacBio technology, we focused on optimizing a 2-step long-range PCR protocol to generate 13-kb amplicons covering the whole FLG exon 3 sequence. The performance of three long-range DNA polymerases was assessed in an attempt to improve the PCR conditions required for the enzymes to function properly. We focused on optimization of the input template DNA concentration and thermocycling parameters to correctly amplify the entire FLG exon 3 sequence, minimizing non-specific amplification.ConclusionsTaken together, our findings suggested that the PrimeSTAR protocol is suitable for producing the amplicons of the 13-kb FLG whole exon 3 to prepare SMRTbell libraries. We suggest that sequencing the generated amplicons may be useful for identifying LoF variants that are causative of the patients' disorders

Male Infertility Diagnosis: Improvement of Genetic Analysis Performance by the Introduction of Pre-Diagnostic Genes in a Next-Generation Sequencing Custom-Made Panel

Background: Infertility affects about 7% of the general male population. The underlying cause of male infertility is undefined in about 50% of cases (idiopathic infertility). The number of genes involved in human spermatogenesis is over two thousand. Therefore, it is essential to analyze a large number of genes that may be involved in male infertility. This study aimed to test idiopathic male infertile patients negative for a validated panel of “diagnostic” genes, for a wide panel of genes that we have defined as “pre-diagnostic.” Methods: We developed a next-generation sequencing (NGS) gene panel including 65 pre-diagnostic genes that were used in 12 patients who were negative to a diagnostic genetic test for male infertility disorders, including primary spermatogenic failure and central hypogonadism, consisting of 110 genes. Results: After NGS sequencing, variants in pre-diagnostic genes were identified in 10/12 patients who were negative to a diagnostic test for primary spermatogenic failure (n = 9) or central hypogonadism (n = 1) due to mutations of single genes. Two pathogenic variants of DNAH5 and CFTR genes and three uncertain significance variants of DNAI1, DNAH11, and CCDC40 genes were found. Moreover, three variants with high impact were found in AMELY, CATSPER 2, and ADCY10 genes. Conclusion: This study suggests that searching for pre-diagnostic genes may be of relevance to find the cause of infertility in patients with apparently idiopathic primary spermatogenic failure due to mutations of single genes and central hypogonadism

Gene variants in Eating Disorders. Focus on Anorexia Nervosa Bulimia Nervosa and Binge-eating Disorder

Eating disorders such as anorexia nervosa, bulimia nervosa and binge-eating disorder, have a deep social impact, concluding with death in cases of severe disease. Eating disorders affect up to 5% of the population in the industrialized countries, but probably the phenomenon is under-detection and under-diagnosis. Eating disorders are multifactorial disorders, resulting from the interaction between environmental triggers, psychological factors, but there is also a strong genetic component. In fact, genetic factors predispose for approximately 33-84% to anorexia nervosa, 28-83% to bulimia nervosa, and 41-57% to binge eating disorder. Twins and family studies have provided an unassailable proof on the heritability of these disorders. Other types of genetic studies, including genome-wide association studies, whole genome sequencing and linkage analysis, allowed to identify the genes and their variants associated with eating disorders and moreover global collaborative efforts have led to delineate the etiology of these disorders. Next Generation Sequencing technologies can be considered as an ideal diagnostic approach to identify not only the common variants, such as single nucleotide polymorphism, but also rare variants. Here we summarize the present knowledge on the molecular etiology and genetic determinants of eating disorders including serotonergic genes, dopaminergic genes, opioid genes, appetite regulation genes, endocannabinoid genes and vitamin D3

Towards a Long-Read Sequencing Approach for the Molecular Diagnosis of RPGRORF15 Genetic Variants

Sequencing of the low-complexity ORF15 exon of RPGR, a gene correlated with retinitis pigmentosa and cone dystrophy, is difficult to achieve with NGS and Sanger sequencing. False results could lead to the inaccurate annotation of genetic variants in dbSNP and ClinVar databases, tools on which HGMD and Ensembl rely, finally resulting in incorrect genetic variants interpretation. This paper aims to propose PacBio sequencing as a feasible method to correctly detect genetic variants in low-complexity regions, such as the ORF15 exon of RPGR, and interpret their pathogenicity by structural studies. Biological samples from 75 patients affected by retinitis pigmentosa or cone dystrophy were analyzed with NGS and repeated with PacBio. The results showed that NGS has a low coverage of the ORF15 region, while PacBio was able to sequence the region of interest and detect eight genetic variants, of which four are likely pathogenic. Furthermore, molecular modeling and dynamics of the RPGR Glu-Gly repeats binding to TTLL5 allowed for the structural evaluation of the variants, providing a way to predict their pathogenicity. Therefore, we propose PacBio sequencing as a standard procedure in diagnostic research for sequencing low-complexity regions such as RPGRORF15, aiding in the correct annotation of genetic variants in online databases

Acute Delta Hepatitis in Italy spanning three decades (1991–2019): Evidence for the effectiveness of the hepatitis B vaccination campaign

Updated incidence data of acute Delta virus hepatitis (HDV) are lacking worldwide. Our aim was to evaluate incidence of and risk factors for acute HDV in Italy after the introduction of the compulsory vaccination against hepatitis B virus (HBV) in 1991. Data were obtained from the National Surveillance System of acute viral hepatitis (SEIEVA). Independent predictors of HDV were assessed by logistic-regression analysis. The incidence of acute HDV per 1-million population declined from 3.2 cases in 1987 to 0.04 in 2019, parallel to that of acute HBV per 100,000 from 10.0 to 0.39 cases during the same period. The median age of cases increased from 27 years in the decade 1991-1999 to 44 years in the decade 2010-2019 (p < .001). Over the same period, the male/female ratio decreased from 3.8 to 2.1, the proportion of coinfections increased from 55% to 75% (p = .003) and that of HBsAg positive acute hepatitis tested for by IgM anti-HDV linearly decreased from 50.1% to 34.1% (p < .001). People born abroad accounted for 24.6% of cases in 2004-2010 and 32.1% in 2011-2019. In the period 2010-2019, risky sexual behaviour (O.R. 4.2; 95%CI: 1.4-12.8) was the sole independent predictor of acute HDV; conversely intravenous drug use was no longer associated (O.R. 1.25; 95%CI: 0.15-10.22) with this. In conclusion, HBV vaccination was an effective measure to control acute HDV. Intravenous drug use is no longer an efficient mode of HDV spread. Testing for IgM-anti HDV is a grey area requiring alert. Acute HDV in foreigners should be monitored in the years to come

Asparagus officinalis (L.): yield and field performance of 10 genotypes cultivated in a semi-arid environment

Asparagus officinalis (L.) (Asparagaceae) is widely cultivated throughout the world, mostly as a traditional food item but, in recent times, also due to its acknowledged many benefits for human health. In Mediterranean environments, this crop proved able to achieve high spear yields, which in many cases may be marketed earlier than competing products, with a significant advantage for local growers. Although the cropping technique of A. officinalis has already been deepened, there is still scarce knowledge about the year-to-year productive stability of the main commercial cultivars in the pedoclimatic conditions of the southern regions of Italy, potentially suitable to the cultivation of this species. This research aimed to provide local farmers with useful information to facilitate the introduction of this species in their farming systems, also giving information about the major factors affecting yield outcomes in these environments. A 3-year cultivation trial was therefore carried out, comparing the productive performances of 10 genotypes of A. officinalis (‘UC157’, ‘Purple’, ‘Italo’, ‘Darbella’, ‘Giove’, ‘Placoseps’, ‘Grande’, ‘Jaleo’, ‘NJ953’ and ‘Atlas’). For experimental purposes, a randomized complete block design was adopted. The results showed that the farming suitability of A. officinalis in the Mediterranean environment is highly variable among cultivars