Functional analysis of three new alpha-thalassemia deletions involving MCS-R2 reveals the presence of an additional enhancer element in the 5' boundary region.

We report three novel deletions involving the Multispecies Conserved Sequences (MCS) R2, also known as the Major Regulative Element (MRE), in patients showing the α-thalassemia phenotype. The three new rearrangements showed peculiar positions of the breakpoints. 1) The (αα)ES is a telomeric 110 kb deletion ending inside the MCS-R3 element. 2) The (αα)FG, 984 bp-long, ends 51 bp upstream to MCS-R2; both are associated with a severe α-thalassemia phenotype. 3) The (αα)CT, 5058 bp-long starts at position +93 of MCS-R2 and is the only one associated to a mild α-thalassemia phenotype. To understand the specific role of different segments of the MCS-R2 element and of its boundary regions we carried out transcriptional and expression analysis. Transcriptional analysis of patients' reticulocytes showed that (αα)ES was unable to produce α2-globin mRNA, while a high level of expression of the α2-globin genes (56%) was detected in (αα)CT deletion, characterized by the presence of the first 93 bp of MCS-R2. Expression analysis of constructs containing breakpoints and boundary regions of the deletions (αα)CT and (αα)FG, showed comparable activity both for MCS-R2 and the boundary region (-682/-8). Considering that the (αα)CT deletion, almost entirely removing MCS-R2, has a less severe phenotype than the (αα)FG α0thalassemia deletion, removing both MCS-R2 almost entirely and an upstream 679 bp, we infer for the first time that an enhancer element must exist in this region that helps to increase the expression of the α-globin genes. The genotype-phenotype relationship of other previously published MCS-R2 deletions strengthened our hypothesis.

Alpha-Thalassemia in Southern Italy: Characterization of Five New Deletions Removing the Alpha-Globin Gene Cluster.

α-thalassemia is characterized in about 80% of cases by deletions generated by the presence of duplications and interspersed repeated sequences in the α-globin gene cluster. In a project on the molecular basis of α-thalassemia in Southern Italy, we identified six families, showing an absence of the most common deletions, and normal α-globin gene sequences. Multiplex Ligation-dependent Probe Amplification (MLPA), qRT-PCR, and the sequencing of long-range PCR amplicon have been used for the identification and characterization of new deletions. MLPA analysis for the identification of α- and ß-globin rearrangement revealed the presence of five new α-thalassemia deletions. The set-up of qRT-PCR allowed us to delimit the extent of the deletions ranging from about 10 kb to more than 250 kb, two of them being of the telomeric type. The long-range PCR generated a specific anomalous fragment in three deletions, and only several unspecific bands in the other two deletions. The sequencing of the anomalous amplicons revealed the breakpoints of two deletions: the --PA, 34 kb long, identified in two families, and the telomeric --AG, 274 kb long. The anomalous fragment containing the breakpoint of the deletion --FG was partially sequenced, and it was not possible to identify the breakpoints due to the presence of several repetitive Alu sequences. The analysis of the breakpoint regions of the --Sciacca and --Puglia, respectively, are about 10 and 165 kb long, and revealed the presence of repeats that most likely impaired the amplification of a specific fragment for the identification of the breakpoint. MLPA, in association with qRT-PCR and long-range PCR, is a good approach for the identification and molecular characterization of rare or new deletions. Breakpoint analysis confirms that Alu sequences play an important role in favoring unequal crossing-over. Southern Italy shows considerable genetic heterogeneity, as expected with its central position in the Mediterranean basin, favoring migratory flows.

Calprotectin Levels and Neutrophil Count Are Prognostic Markers of Mortality in COVID-19 Patients.

Inflammation plays a crucial role in worsening coronavirus disease (COVID-19). Calprotectin is a pro-inflammatory molecule produced by monocytes and neutrophilic granulocytes. The aim of the study was to evaluate both the prognostic role of circulating calprotectin levels and neutrophil count toward fatal outcome in COVID-19 patients. We retrospectively collected and analyzed data on 195 COVID-19 adult patients, 156 hospitalized in the infectious disease unit and 39 in the intensive care unit (ICU). Calprotectin levels and neutrophil counts measured at the first hospitalization day were higher in the patients with a fatal outcome than in surviving ones. The association of high calprotectin levels and neutrophil count to patient death remain significant by logistic regression, independent of patient age. ROC curves analysis for calprotectin levels and neutrophil count revealed a good discriminatory power toward survival (area under the curve of 0.759 and 0.843, respectively) and identified the best cut-off (1.66 mg/L and 16.39 × 103/µL, respectively). Kaplan-Meier analysis confirmed the prognostic role of high calprotectin levels and neutrophil count in death prediction. In conclusion, this study highlights that calprotectin levels together with neutrophil count should be considered as biomarkers of mortality in COVID-19 patients.

mRNA Analysis of Frameshift Mutations with Stop Codon in the Last Exon: The Case of Hemoglobins Campania [α1 cod95 (-C)] and Sciacca [α1 cod109 (-C)].

An insertion or deletion of a nucleotide (nt) in the penultimate or the last exon can result in a frameshift and premature termination codon (PTC), giving rise to an unstable protein variant, showing a dominant phenotype. We described two α-globin mutants created by the deletion of a nucleotide in the penultimate or the last exon of the α1-globin gene: the Hb Campania or α1 cod95 (-C), causing a frameshift resulting in a PTC at codon 102, and the Hb Sciacca or α1 cod109 (-C), causing a frameshift and formation of a PTC at codon 133. The carriers showed α-thalassemia alterations (mild microcytosis with normal Hb A2) and lacked hemoglobin variants. The 3D model indicated the α-chain variants' instability, due to the severe structural alterations with impairment of the chaperone alpha-hemoglobin stabilizing protein (AHSP) interaction. The qualitative and semiquantitative analyses of the α1mRNA from the reticulocytes of carriers highlighted a reduction in the variant cDNAs that constituted 34% (Hb Campania) and 15% (Hb Sciacca) of the total α1-globin cDNA, respectively. We developed a workflow for the in silico analysis of mechanisms triggering no-go decay, and its results suggested that the reduction in the variant mRNA was likely due to no-go decay caused by the presence of a rare triplet, and, in the case of Hb Sciacca, also by the mRNA's secondary structure variation. It would be interesting to correlate the phenotype with the quantity of other frameshift mRNA variants, but very few data concerning α- and ß-globin variants are available.

Effect of Mutations on mRNA and Globin Stability: The Cases of Hb Bernalda/Groene Hart and Hb Southern Italy.

We identified two unstable variants in the third exon of α-globin genes: Hb Bernalda/Groene Hart (HBA1:c.358C>T), and Hb Caserta (HBA2:c.79G>A) in cis to Hb Sun Prairie (HBA2:c.391G>C), also named Hb Southern Italy. These mutations occurred in the H helix of the α-globin that is involved in heme contacting, specific recognition of α-hemoglobin-stabilizing protein (AHSP), and α1ß1 interactions. The carriers showed α-thalassemia phenotype, but one also jaundice and cholelithiasis. Molecular identification of clusters of families in Southern Italy encouraged molecular characterization of mRNA, globin chain analyses, molecular modeling studies, and comparison with globin variants to understand the mechanisms causing the α-thalassemia phenotype. A normal amount of Hb Bernalda/Groene Hart mRNA were found, and molecular modeling highlighted additional H bonds with AHSP. For Hb Southern Italy, showing an unexpected α/ß biosynthetic ratio typical of the ß-thalassemia type, two different molecular mechanisms were shown: Reduction of the variant mRNA, likely due to the No-Go Decay for the presence of unused triplet ACG at cod 26, and protein instability due to the impairment of AHSP interaction. The UDP glucuronosyltransferase 1A (UGT1A1) genotyping was conclusive in the case of jaundice and cholelithiasis. Multiple approaches are needed to properly identify the mechanisms leading to unstable variants and the effect of a mutation.

WTAP and BIRC3 are involved in the posttranscriptional mechanisms that impact on the expression and activity of the human lactonase PON2.

The activity of human paraoxonase 2 (PON2) is rapidly reduced in cells incubated with the bacterial quorormone 3-Oxo-dodecanoyl Homoserine Lactone (3OC12HSL), an observation that led to hypothesize a fast PON2 post-translational modification (PTM). Recently, we detected a 3OC12HSL-induced PTM in a cell-free system in which a crude extract from 3OC12HSL-treated HeLa cells was able to inactivate and ubiquitinate at position 144 a recombinant PON2. Here we show the occurrence of this and new PTMs on PON2 in HeLa cells. PTMs were found to gather nearby the two SNPs, A148G, and S311C, that are related to type-2 diabetes and its complications. Furthermore, we detected a PTM nearby a 12 amino acids region that is deleted in PON2 Isoform 2. An in vitro mutation analysis showed that the SNPs and the deletion are involved in PON2 activity and suggested a role of PTMs on its modulation, while a SAXS analysis pointed to Isoform 2 as being largely unstructured, compared to the wild type. Besides, we discovered a control of PON2 expression via a putative mRNA operon involving the Wilms tumor 1 associated protein (WTAP) and the E3 ubiquitin ligase (E3UbL) baculoviral IAP repeat-containing 3 (BIRC3).

Role of nonsense-mediated decay and nonsense-associated altered splicing in the mRNA pattern of two new α-thalassemia mutants.

α-thalassemia is a common disease characterized mainly by deletion mutants. We identified two new α-thalassemia pointform mutants: α1cod22 GGC>GGT Gly>Gly creating a 5' splicing sequence and α1cod23 GAG>TAG Glu>stop. We performed qualitative and semi-quantitative analysis of the mRNA molecules, from carriers' blood, to define the molecular mechanisms giving rise to the thalassemia phenotype. In vitro analysis using α-globin constructs and cycloheximide was performed to evaluate if the mutants are substrates of nonsense-mediated mRNA decay (NMD). In the α1cod22 GGC>GGT the new 5' splicing site in exon 1 completely substitutes the normal one. We demonstrated the presence of mRNA decay as the abnormally spliced mRNA was consistent in the nucleus, partially degraded in the cytoplasm of cultured cells, but only 2.8% in the reticulocytes. The analysis of the αcod23 transcript showed an escape from the NMD as for the human ß-globin transcript with nonsense mutations in the first exon: the anomalous mRNA was reduced in the nucleus, followed by only a slight lowering from 32% to 27% of the normal α1 mRNA in the reticulocytes. In both the mutants we showed a moderate sensitivity to the NMD assay and we speculate the activation of other RNA surveillance mechanisms for the αcod22 mutant. No activation of cryptic splice sites was detected and no role could be assigned to the nonsense-associated altered splicing. Studies on transcripts from patient cells represent a very useful approach providing considerable information about the processes occuring in vivo.

Identification and molecular characterization of a novel 163 kb deletion: The Italian (ϵγδß)(0)-thalassemia.

OBJECTIVE AND IMPORTANCE: To verify the presence of ß-thalassemia in subjects showing hematologic phenotype of α-thalassemia, conduct normal molecular sequence analysis of the α-globin genes, and detect the absence of the most frequent α-thalassemia deletions. CLINICAL PRESENTATION: A patient from Apulia (Southern Italy) was referred to our institution for the occasional founding of hypochromic polyglobulia and microcytic red blood cells associated with normal levels of Hb A2 and Hb F and normal iron parameters. INTERVENTION AND TECHNIQUE: The patient has been investigated using Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), quantitative real-time PCR, restriction analysis, and gap-PCR. A novel deletion, the Italian (ϵγδß)(0)-thalassemia, has been identified. The 5' breakpoint was within a LINE element of 80 kb 3' of the ε-globin gene, and the 3' breakpoint was within a 160-bp palindrome of about 30 kb 5' of the ß-globin gene. The breakpoint region was characterized by the presence of a microhomology (5'-TCT-3') and of an insertion of 43 bp owing to the duplication of the 160-bp palindrome. Comparison of the Hb and Hb A2 values of (ϵγδß)(0)-thalassemia from the literature with those of (molecularly known) thalassemia carriers indicated a higher level of Hb A2 with respect to α-thalassemia and a lower level of Hb with respect to ß(0)-thalassemia carriers. CONCLUSION: In this study, we report the first (ϵγδß)(0)-thalassemia case identified in Italy. To avoid misdiagnosis of ß-thalassemia, we suggest verifying the presence of large deletions of the ß-globin gene cluster in subjects showing a higher border line level of Hb A2 and a lower level of Hb.

α-Thalassemia associated with hb instability: a tale of two features. the case of Hb Rogliano or α1 Cod 108(G15)Thr→Asn and Hb Policoro or α2 Cod 124(H7)Ser→Pro.

We identified two new variants in the third exon of the α-globin gene in families from southern Italy: the Hb Rogliano, α1 cod108 ACC>AAC or α1[α108(G15)Thr→Asn] and the Hb Policoro, α2 cod124 TCC>CCC or α2[α124(H7)Ser→Pro]. The carriers showed mild α-thalassemia phenotype and abnormal hemoglobin stability features. These mutations occurred in the G and H helices of the α-globin both involved in the specific recognition of AHSP and ß1 chain. Molecular characterization of mRNA, globin chain analyses and molecular modelling studies were carried out to highlight the mechanisms causing the α-thalassemia phenotype. The results demonstrated that the α-thalassemia defect associated with the two Hb variants originated by different defects. Hb Rogliano showed an intrinsic instability of the tetramer due to anomalous intra- and inter-chain interactions suggesting that the variant chain is normally synthesized and complexed with AHSP but rapidly degraded because it is unable to form the α1ß1 dimers. On the contrary in the case of Hb Policoro two different molecular mechanisms were shown: the reduction of the variant mRNA level by an unclear mechanism and the protein instability due to impairment of AHSP interaction. These data highlighted that multiple approaches, including mRNA quantification, are needed to properly identify the mechanisms leading to the α-thalassemia defect. Elucidation of the specific mechanism leads to the definition of a given phenotype providing important guidance for the diagnosis of unstable variants.