The different types of land-use and soil lithology in urban and peri-urban areas of modern cities compose a complex mosaic of soil ecosystems. It is largely unknown how these differences result in ...changes in bacterial community composition and structure as well as in functional guilds involved in N cycling. To investigate the bacterial composition and the proportion of denitrifiers in agricultural, forested, schoolyard and industrial areas, 24 samples were collected from urban and peri-urban sites of Lefkosia. Bacterial diversity and the proportion of denitrifiers were assessed by NGS and qPCR, respectively. Proteobacteria, Actinobacteria, Bacteriodetes, Chloroflexi, Acidobacteria and Planctomycetes were identified as the most dominant phyla across all sites, while agricultural sites exhibited the highest bacterial diversity. Heavy metals such as Co, Pb, V and Al were identified as key factors shaping bacterial composition in industrial and schoolyard sites, while the bacterial assemblages in agricultural and forested sites were associated with Ca. Variance partitioning analysis showed that 10.2% of the bacterial community variation was explained by land use management, 5.1% by chemical elements due to soil lithology, and 1.4% by sampling location. The proportion of denitrifiers varied with land use management. In industrial and schoolyard sites, the abundance of the nosZII bacterial community increased while nirK abundance declined. Our data showed that land use and lithology have a moderate impact on the bacterial assemblages in urban and peri-urban areas of Lefkosia. As the nosZII bacterial community is important to the N
O sink capacity of soils, it would be interesting to elucidate the factors contributing to the proliferation of the nosZII clade in these soils.
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Several types of haemoglobinopathies are caused by copy number variants (CNVs). While diagnosis is often based on haematological and biochemical parameters, a definitive diagnosis requires molecular ...DNA analysis. In some cases, the molecular characterisation of large deletions/duplications is challenging and inconclusive and often requires the use of specific diagnostic procedures, such as multiplex ligation-dependent probe amplification (MLPA). Herein, we collected and comprehensively analysed all known CNVs associated with haemoglobinopathies. The dataset of 291 CNVs was retrieved from the IthaGenes database and was further manually annotated to specify genomic locations, breakpoints and MLPA probes relevant for each CNV. We developed IthaCNVs, a publicly available and easy-to-use online tool that can facilitate the diagnosis of rare and diagnostically challenging haemoglobinopathy cases attributed to CNVs. Importantly, it facilitates the filtering of available entries based on the type of breakpoint information, on specific chromosomal and locus positions, on MLPA probes, and on affected gene(s). IthaCNVs brings together manually curated information about CNV genomic locations, functional effects, and information that can facilitate CNV characterisation through MLPA. It can help laboratory staff and clinicians confirm suspected diagnosis of CNVs based on molecular DNA screening and analysis.
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Haemoglobinopathies are the commonest monogenic diseases worldwide and are caused by variants in the globin gene clusters. With over 2400 variants detected to date, their interpretation using the ...American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines is challenging and computational evidence can provide valuable input about their functional annotation. While many in silico predictors have already been developed, their performance varies for different genes and diseases. In this study, we evaluate 31 in silico predictors using a dataset of 1627 variants in
,
and
. By varying the decision threshold for each tool, we analyse their performance (a) as binary classifiers of pathogenicity and (b) by using different non-overlapping pathogenic and benign thresholds for their optimal use in the ACMG/AMP framework. Our results show that CADD, Eigen-PC, and REVEL are the overall top performers, with the former reaching moderate strength level for pathogenic prediction. Eigen-PC and REVEL achieve the highest accuracies for missense variants, while CADD is also a reliable predictor of non-missense variants. Moreover, SpliceAI is the top performing splicing predictor, reaching strong level of evidence, while GERP++ and phyloP are the most accurate conservation tools. This study provides evidence about the optimal use of computational tools in globin gene clusters under the ACMG/AMP framework.
The +33 C>G variant NM_000518.5(HBB):c.-18C>G in the 5' untranslated region (UTR) of the β-globin gene is described in the literature as both mild and silent, while it causes a phenotype of ...thalassemia intermedia in the presence of a severe β-thalassemia allele. Despite its potential clinical significance, the determination of its pathogenicity according to established standards requires a greater number of published cases and co-segregation evidence than what is currently available. The present study provides an extensive phenotypic characterization of +33 C>G using 26 heterozygous and 11 compound heterozygous novel cases detected in Cyprus and employs computational predictors (CADD, RegulomeDB) to better understand its impact on clinical severity. Genotype identification of globin gene variants, including α- and δ-thalassemia determinants, and rs7482144 (XmnI) was carried out using Sanger sequencing, gap-PCR, and restriction enzyme digestion methods. The heterozygous state of +33 C>G had a silent phenotype without apparent microcytosis or hypochromia, while compound heterozygosity with a β+ or β0 allele had a spectrum of clinical phenotypes. Awareness of the +33 C>G is required across Mediterranean populations where β-thalassemia is frequent, particularly in Cyprus, with significant relevance in population screening and fetal diagnostic applications.
Haemoglobinopathies are common monogenic disorders with diverse clinical manifestations, partly attributed to the influence of modifier genes. Recent years have seen enormous growth in the amount of ...genetic data, instigating the need for ranking methods to identify candidate genes with strong modifying effects. Here, we present the first evidence-based gene ranking metric (IthaScore) for haemoglobinopathy-specific phenotypes by utilising curated data in the IthaGenes database. IthaScore successfully reflects current knowledge for well-established disease modifiers, while it can be dynamically updated with emerging evidence. Protein-protein interaction (PPI) network analysis and functional enrichment analysis were employed to identify new potential disease modifiers and to evaluate the biological profiles of selected phenotypes. The most relevant gene ontology (GO) and pathway gene annotations for (a) haemoglobin (Hb) F levels/Hb F response to hydroxyurea included urea cycle, arginine metabolism and vascular endothelial growth factor receptor (VEGFR) signalling, (b) response to iron chelators included xenobiotic metabolism and glucuronidation, and (c) stroke included cytokine signalling and inflammatory reactions. Our findings demonstrate the capacity of IthaGenes, together with dynamic gene ranking, to expand knowledge on the genetic and molecular basis of phenotypic variation in haemoglobinopathies and to identify additional candidate genes to potentially inform and improve diagnosis, prognosis and therapeutic management.
Therapy via the gene addition of the anti-sickling βAS3-globin transgene is potentially curative for all β-hemoglobinopathies and therefore of particular clinical and commercial interest. This study ...investigates GLOBE-based lentiviral vectors (LVs) for βAS3-globin addition and evaluates strategies for an increased β-like globin expression without vector dose escalation. First, we report the development of a GLOBE-derived LV, GLV2-βAS3, which, compared to its parental vector, adds anti-sickling action and a transcription-enhancing 848-bp transcription terminator element, retains high vector titers and allows for superior β-like globin expression in primary patient-derived hematopoietic stem and progenitor cells (HSPCs). Second, prompted by our previous correction of HBBIVSI−110(G>A) thalassemia based on RNApol(III)-driven shRNAs in mono- and combination therapy, we analyzed a series of novel LVs for the RNApol(II)-driven constitutive or late-erythroid expression of HBBIVSI−110(G>A)-specific miRNA30-embedded shRNAs (shRNAmiR). This included bifunctional LVs, allowing for concurrent βAS3-globin expression. LVs were initially compared for their ability to achieve high β-like globin expression in HBBIVSI−110(G>A)-transgenic cells, before the evaluation of shortlisted candidate LVs in HBBIVSI−110(G>A)-homozygous HSPCs. The latter revealed that β-globin promoter-driven designs for monotherapy with HBBIVSI−110(G>A)-specific shRNAmiRs only marginally increased β-globin levels compared to untransduced cells, whereas bifunctional LVs combining miR30-shRNA with βAS3-globin expression showed disease correction similar to that achieved by the parental GLV2-βAS3 vector. Our results establish the feasibility of high titers for LVs containing the full HBB transcription terminator, emphasize the importance of the HBB terminator for the high-level expression of HBB-like transgenes, qualify the therapeutic utility of late-erythroid HBBIVSI−110(G>A)-specific miR30-shRNA expression and highlight the exceptional potential of GLV2-βAS3 for the treatment of severe β-hemoglobinopathies.
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