Production and Characterization of K562 Cellular Clones Hyper-Expressing the Gene Encoding α-Globin: Preliminary Analysis of Biomarkers Associated with Autophagy.

One of the most relevant pathophysiological hallmarks of ß-thalassemia is the accumulation of toxic α-globin chains inside erythroid cells, which is responsible for their premature death (hemolysis). In this context, the availability of an experimental model system mimicking the excess in α-globin chain production is still lacking. The objective of the present study was to produce and characterize K562 cellular clones forced to produce high amounts of α-globin, in order to develop an experimental model system suitable for studies aimed at the reduction of the accumulation of toxic α-globin aggregates. In the present study, we produced and characterized K562 cellular clones that, unlike the original K562 cell line, stably produced high levels of α-globin protein. As expected, the obtained clones had a tendency to undergo apoptosis that was proportional to the accumulation of α-globin, confirming the pivotal role of α-globin accumulation in damaging erythroid cells. Interestingly, the obtained clones seemed to trigger autophagy spontaneously, probably to overcome the accumulation/toxicity of the α-globin. We propose this new model system for the screening of pharmacological agents able to activate the full program of autophagy to reduce α-globin accumulation, but the model may be also suitable for new therapeutical approaches targeted at the reduction of the expression of the α-globin gene.

The rs368698783 (G>A) Polymorphism Affecting LYAR Binding to the Aγ-Globin Gene Is Associated with High Fetal Hemoglobin (HbF) in ß-Thalassemia Erythroid Precursor Cells Treated with HbF Inducers

The human homologue of mouse Ly-1 antibody reactive clone protein (LYAR) is a putative novel regulator of γ-globin gene transcription. The LYAR DNA-binding motif (5'-GGTTAT-3') is located within the 5'-UTR of the Aγ-globin gene. The LYAR rs368698783 (G>A) polymorphism is present in ß-thalassemia patients and decreases the LYAR binding efficiency to the Aγ-globin gene. The objective of this study was to stratify ß-thalassemia patients with respect to the rs368698783 (G>A) polymorphism and to verify whether their erythroid precursor cells (ErPCs) differentially respond in vitro to selected fetal hemoglobin (HbF) inducers. The rs368698783 (G>A) polymorphism was detected by DNA sequencing, hemoglobin production by HPLC, and accumulation of globin mRNAs by RT-qPCR. We found that the LYAR rs368698783 (G>A) polymorphism is associated with high basal and induced production of fetal hemoglobin in ß-thalassemia patients. The most striking association was found using rapamycin as an HbF inducer. The results presented here could be considered important not only for basic biomedicine but also in applied translational research for precision medicine in personalized therapy of ß-thalassemia. Accordingly, our data suggest that the rs368698783 polymorphism might be considered among the parameters useful to recruit patients with the highest probability of responding to in vivo hydroxyurea (HU) treatment.

Expression of γ-globin genes in ß-thalassemia patients treated with sirolimus: results from a pilot clinical trial (Sirthalaclin).

Introduction: ß-thalassemia is caused by autosomal mutations in the ß-globin gene, which induce the absence or low-level synthesis of ß-globin in erythroid cells. It is widely accepted that a high production of fetal hemoglobin (HbF) is beneficial for patients with ß-thalassemia. Sirolimus, also known as rapamycin, is a lipophilic macrolide isolated from a strain of Streptomyces hygroscopicus that serves as a strong HbF inducer in vitro and in vivo. In this study, we report biochemical, molecular, and clinical results of a sirolimus-based NCT03877809 clinical trial (a personalized medicine approach for ß-thalassemia transfusion-dependent patients: testing sirolimus in a first pilot clinical trial, Sirthalaclin). Methods: Accumulation of γ-globin mRNA was analyzed using reverse-transcription quantitative polymerase chain reaction (PCR), while the hemoglobin pattern was analyzed using high-performance liquid chromatography (HPLC). The immunophenotype was analyzed using a fluorescence-activated cell sorter (FACS), with antibodies against CD3, CD4, CD8, CD14, CD19, CD25 (for analysis of peripheral blood mononuclear cells), or CD71 and CD235a (for analysis of in vitro cultured erythroid precursors). Results: The results were obtained in eight patients with the ß+/ß+ and ß+/ß0 genotypes, who were treated with a starting dosage of 1 mg/day sirolimus for 24-48 weeks. The first finding of this study was that the expression of γ-globin mRNA increased in the blood and erythroid precursor cells isolated from ß-thalassemia patients treated with low-dose sirolimus. This trial also led to the important finding that sirolimus influences erythropoiesis and reduces biochemical markers associated with ineffective erythropoiesis (excess free α-globin chains, bilirubin, soluble transferrin receptor, and ferritin). A decrease in the transfusion demand index was observed in most (7/8) of the patients. The drug was well tolerated, with minor effects on the immunophenotype, and an only side effect of frequently occurring stomatitis. Conclusion: The data obtained indicate that low doses of sirolimus modify hematopoiesis and induce increased expression of γ-globin genes in a subset of patients with ß-thalassemia. Further clinical trials are warranted, possibly including testing of the drug in patients with less severe forms of the disease and exploring combination therapies.

Teaching during COVID-19 pandemic in practical laboratory classes of applied biochemistry and pharmacology: A validated fast and simple protocol for detection of SARS-CoV-2 Spike sequences.

The pandemic caused by the SARS-CoV-2 virus (COVID-19) is still a major health issue. The COVID-19 pandemic has forced the university teaching to consider in high priority the switch from in-presence teaching to remote teaching, including laboratory teaching. While excellent virtual-laboratory teaching has been proposed and turned out to be very useful, the need of a real-laboratory in-presence teaching is still a major need. This study was aimed at presenting a laboratory exercise focusing (a) on a very challenging therapeutic strategy, i.e. SARS-CoV-2 diagnostics, and (b) on technologies that are playing a central role in applied biochemistry and molecular biology, i.e. PCR and RT-PCR. The aims of the practical laboratory were to determine: (a) the possibility to identify SARS-CoV-2 sequences starting from a recombinant plasmid and (b) the possibility to discriminate cells with respect to the expression of SARS-CoV-2 Spike protein. This activity is simple (cell culture, RNA extraction, RT-qPCR are all well-established technologies), fast (starting from isolated and characterized RNA, few hours are just necessary), highly reproducible (therefore easily employed by even untrained students). We suggest that this laboratory practical exercises should be considered for face-to-face teaching especially if the emergency related to the COVID-19 pandemic is maintained. The teaching protocol here described might be considered in order to perform fast but meaningful in-presence teaching, making feasible the division of crowded classes in low-number cohorts of students, allowing the maintenance of the required social distance.

Droplet Digital PCR for Non-Invasive Prenatal Detection of Fetal Single-Gene Point Mutations in Maternal Plasma.

Non-invasive prenatal testing (NIPT) is based on the detection and characterization of circulating cell-free fetal DNA (ccffDNA) in maternal plasma and aims to identify genetic abnormalities. At present, commercial NIPT kits can detect only aneuploidies, small deletions and insertions and some paternally inherited single-gene point mutations causing genetic diseases, but not maternally inherited ones. In this work, we have developed two NIPT assays, based on the innovative and sensitive droplet digital PCR (ddPCR) technology, to identify the two most common ß thalassemia mutations in the Mediterranean area (ß+IVSI-110 and ß039), maternally and/or paternally inherited, by fetal genotyping. The assays were optimized in terms of amplification efficiency and hybridization specificity, using mixtures of two genomic DNAs with different genotypes and percentages to simulate fetal and maternal circulating cell-free DNA (ccfDNA) at various gestational weeks. The two ddPCR assays were then applied to determine the fetal genotype from 52 maternal plasma samples at different gestational ages. The diagnostic outcomes were confirmed for all the samples by DNA sequencing. In the case of mutations inherited from the mother or from both parents, a precise dosage of normal and mutated alleles was required to determine the fetal genotype. In particular, we identified two diagnostic ranges for allelic ratio values statistically distinct and not overlapping, allowing correct fetal genotype determinations for almost all the analyzed samples. In conclusion, we have developed a simple and sensitive diagnostic tool, based on ddPCR, for the NIPT of ß+IVSI-110 and ß039 mutations paternally and, for the first time, maternally inherited, a tool, which may be applied to other single point mutations causing monogenic diseases.

Nanoparticle-Enhanced Surface Plasmon Resonance Imaging Enables the Ultrasensitive Detection of Non-Amplified Cell-Free Fetal DNA for Non-Invasive Prenatal Testing.

Although many potential applications in early clinical diagnosis have been proposed, the use of a surface plasmon resonance imaging (SPRI) technique for non-invasive prenatal diagnostic approaches based on maternal blood analysis is confined. Here, we report a nanoparticle-enhanced SPRI strategy for a non-invasive prenatal fetal sex determination based on the detection of a Y-chromosome specific sequence (single-gene SRY) in cell-free fetal DNA from maternal plasma. The SPR assay proposed here allows for detection of male DNA in mixtures of 2.5 aM male and female genomic DNAs with no preliminary amplification of the DNA target sequence, thus establishing an analytical protocol that does not require costly, time-consuming, and prone to sample contamination PCR-based procedures. Afterward, the developed protocol was successfully applied to reveal male cell-free fetal DNA in the plasma of pregnant women at different gestational ages, including early gestational ages. This approach would pave the way for the establishment of faster and cost-effective non-invasive prenatal testing.

In vitro induction of interleukin-8 by SARS-CoV-2 Spike protein is inhibited in bronchial epithelial IB3-1 cells by a miR-93-5p agomiR.

One of the major clinical features of COVID-19 is a hyperinflammatory state, which is characterized by high expression of cytokines (such as IL-6 and TNF-α), chemokines (such as IL-8) and growth factors and is associated with severe forms of COVID-19. For this reason, the control of the "cytokine storm" represents a key issue in the management of COVID-19 patients. In this study we report evidence that the release of key proteins of the COVID-19 "cytokine storm" can be inhibited by mimicking the biological activity of microRNAs. The major focus of this report is on IL-8, whose expression can be modified by the employment of a molecule mimicking miR-93-5p, which is able to target the IL-8 RNA transcript and modulate its activity. The results obtained demonstrate that the production of IL-8 protein is enhanced in bronchial epithelial IB3-1 cells by treatment with the SARS-CoV-2 Spike protein and that IL-8 synthesis and extracellular release can be strongly reduced using an agomiR molecule mimicking miR-93-5p.

Discovery of Novel Fetal Hemoglobin Inducers through Small Chemical Library Screening.

The screening of chemical libraries based on cellular biosensors is a useful approach to identify new hits for novel therapeutic targets involved in rare genetic pathologies, such as ß-thalassemia and sickle cell disease. In particular, pharmacologically mediated stimulation of human γ-globin gene expression, and increase of fetal hemoglobin (HbF) production, have been suggested as potential therapeutic strategies for these hemoglobinopathies. In this article, we screened a small chemical library, constituted of 150 compounds, using the cellular biosensor K562.GR, carrying enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes under the control of the human γ-globin and ß-globin gene promoters, respectively. Then the identified compounds were analyzed as HbF inducers on primary cell cultures, obtained from ß-thalassemia patients, confirming their activity as HbF inducers, and suggesting these molecules as lead compounds for further chemical and biological investigations.

mTOR and STAT3 Pathway Hyper-Activation is Associated with Elevated Interleukin-6 Levels in Patients with Shwachman-Diamond Syndrome: Further Evidence of Lymphoid Lineage Impairment.

Shwachman-Diamond syndrome (SDS) is a rare inherited bone marrow failure syndrome, resulting in neutropenia and a risk of myeloid neoplasia. A mutation in a ribosome maturation factor accounts for almost all of the cases. Lymphoid involvement in SDS has not been well characterized. We recently reported that lymphocyte subpopulations are reduced in SDS patients. We have also shown that the mTOR-STAT3 pathway is hyper-activated in SDS myeloid cell populations. Here we show that mTOR-STAT3 signaling is markedly upregulated in the lymphoid compartment of SDS patients. Furthermore, our data reveal elevated IL-6 levels in cellular supernatants obtained from lymphoblasts, bone marrow mononuclear and mesenchymal stromal cells, and plasma samples obtained from a cohort of 10 patients. Of note, everolimus-mediated inhibition of mTOR signaling is associated with basal state of phosphorylated STAT3. Finally, inhibition of mTOR-STAT3 pathway activation leads to normalization of IL-6 expression in SDS cells. Altogether, our data strengthen the hypothesis that SDS affects both lymphoid and myeloid blood compartment and suggest everolimus as a potential therapeutic agent to reduce excessive mTOR-STAT3 activation in SDS.

Detection of the sickle hemoglobin allele using a surface plasmon resonance based biosensor.

Sickle Cell Disease (SCD) is a monogenic hereditary blood disorder caused by a single point mutation (ßS) in the ß globin gene resulting in an abnormal hemoglobin (HbS) that can polymerize within the erythrocytes, inducing their characteristic sickle shape. This causes hemolytic anemia and occlusive vessels for the most severe clinical status. Molecular analysis is crucial for fast and precise diagnosis of different forms of SCD, and, on the basis of underlying genotype, for supporting the most appropriate treatment options. In this context, we describe a simple and reproducible protocol for the molecular identification of the ßS mutation based on surface plasmon resonance (SPR) using the Biacore™ X100 affinity biosensor. This technology has already demonstrated its diagnostic suitability for the identification of point mutations responsible for genetic diseases such as cystic fibrosis and ß thalassemia, using a protocol based on immobilization of PCR products on the sensor chip. On the contrary, in this work we applied a SPR strategy based on an innovative interaction format, recently developed in our group also for ß thalassemia mutations. In particular, we correctly detected the ßS mutation responsible for SCD, both in homozygous and heterozygous states, after hybridization of two oligonucleotide probes (normal and mutated) for the ßS mutation, immobilized on sensor chip, with unbalanced PCR products obtained from 53 genomic DNAs carrying different ßS allele combinations.

Surface plasmon resonance based analysis of the binding of LYAR protein to the rs368698783 (G>A) polymorphic Aγ-globin gene sequences mutated in ß-thalassemia.

Recent studies have identified and characterized a novel putative transcriptional repressor site in a 5' untranslated region of the Aγ-globin gene that interacts with the Ly-1 antibody reactive clone (LYAR) protein. LYAR binds the 5'-GGTTAT-3' site of the Aγ-globin gene, and this molecular interaction causes repression of gene transcription. In ß-thalassemia patients, a polymorphism has been demonstrated (the rs368698783 G>A polymorphism) within the 5'-GGTTAT-3' LYAR-binding site of the Aγ-globin gene. The major results gathered from surface plasmon resonance based biospecific interaction analysis (SPR-BIA) studies (using crude nuclear extracts, LYAR-enriched lysates, and recombinant LYAR) support the concept that the rs368698783 G>A polymorphism of the Aγ-globin gene attenuates the efficiency of LYAR binding to the LYAR-binding site. This conclusion was fully confirmed by a molecular docking analysis. This might lead to a very important difference in erythroid cells from ß-thalassemia patients in respect to basal and induced levels of production of fetal hemoglobin. The novelty of the reported SPR-BIA method is that it allows the characterization and validation of the altered binding of a key nuclear factor (LYAR) to mutated LYAR-binding sites. These results, in addition to theoretical implications, should be considered of interest in applied pharmacology studies as a basis for the screening of drugs able to inhibit LYAR-DNA interactions. This might lead to the identification of molecules facilitating induced increase of γ-globin gene expression and fetal hemoglobin production in erythroid cells, which is associated with possible reduction of the clinical severity of the ß-thalassemia phenotype. Graphical abstract.

Development and characterization of cellular biosensors for HTS of erythroid differentiation inducers targeting the transcriptional activity of γ-globin and ß-globin gene promoters.

There is a general agreement that pharmacologically mediated stimulation of human γ-globin gene expression and increase of production of fetal hemoglobin (HbF) is a potential therapeutic approach in the experimental therapy of ß-thalassemia and sickle cell anemia. Here, we report the development and characterization of cellular biosensors carrying enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes under the control of the human γ-globin and ß-globin gene promoters, respectively; these dual-reporter cell lines are suitable to identify the induction ability of screened compounds on the transcription in erythroid cells of γ-globin and ß-globin genes by FACS with efficiency and reproducibility. Our experimental system allows to identify (a) HbF inducers stimulating to different extent the activity of the γ-globin gene promoter and (b) molecules that stimulate also the activity of the ß-globin gene promoter. A good correlation does exist between the results obtained by using the EGFP/RFP clones and experiments performed on erythroid precursor cells from ß-thalassemic patients, confirming that this experimental system can be employed for high-throughput screening (HTS) analysis. Finally, we have demonstrated that this dual-reporter cell line can be used for HTS in 384-well plate, in order to identify novel HbF inducers for the therapy of ß-thalassemia and sickle cell anemia. Graphical abstract.

Non-invasive Prenatal Testing Using Fetal DNA.

Non-invasive prenatal diagnosis (NIPD) is based on fetal DNA analysis starting from a simple peripheral blood sample, thus avoiding risks associated with conventional invasive techniques. During pregnancy, the fetal DNA increases to approximately 3-13% of the total circulating free DNA in maternal plasma. The very low amount of circulating cell-free fetal DNA (ccffDNA) in maternal plasma is a crucial issue, and requires specific and optimized techniques for ccffDNA purification from maternal plasma. In addition, highly sensitive detection approaches are required. In recent years, advanced ccffDNA investigation approaches have allowed the application of non-invasive prenatal testing (NIPT) to determine fetal sex, fetal rhesus D (RhD) genotyping, aneuploidies, micro-deletions and the detection of paternally inherited monogenic disorders. Finally, complex and innovative technologies such as digital polymerase chain reaction (dPCR) and next-generation sequencing (NGS) (exhibiting higher sensitivity and/or the capability to read the entire fetal genome from maternal plasma DNA) are expected to allow the detection, in the near future, of maternally inherited mutations that cause genetic diseases. The aim of this review is to introduce the principal ccffDNA characteristics and their applications as the basis of current and novel NIPT.

Non-invasive fetal sex diagnosis in plasma of early weeks pregnants using droplet digital PCR.

BACKGROUND: Fetal sex determination is useful for families at risk of X-linked disorders, such as Duchenne muscular dystrophy, adrenal hypoplasia, hemophilia. At first, this could be obtained through invasive procedures such as amniocentesis and chorionic villus sampling, having a 1% risk of miscarriage. Since the discovery of cell-free fetal DNA (cffDNA) in maternal plasma, noninvasive prenatal testing permits the early diagnosis of fetal sex through analysis of cffDNA. However, the low amount of cffDNA relative to circulating maternal DNA requires highly sensitive molecular techniques in order to perform noninvasive prenatal diagnosis. In this context we employed droplet digital PCR (ddPCR) in order to evaluate the earliest possible fetal sex determination from circulating DNA extracted from plasma of pregnant women at different gestational ages. METHODS: We identified the fetal sex on cffDNA extracted from 29 maternal plasma samples at early gestational ages, several of them not suitable for qPCR determination, using ddPCR designed for SRY gene target. RESULTS: All maternal plasma samples were determined correctly for SRY gene target using ddPCR even at very early gestational age (prior to 7 weeks). CONCLUSIONS: The ddPCR is a robust, efficient and reliable technology for the earliest possible fetal sex determination from maternal plasma.

UPF1 silenced cellular model systems for screening of read-through agents active on ß039 thalassemia point mutation.

BACKGROUND: Nonsense mutations promote premature translational termination, introducing stop codons within the coding region of mRNAs and causing inherited diseases, including thalassemia. For instance, in ß039 thalassemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, ribosomal read-through molecules, such as aminoglycoside antibiotics, have been tested on mRNAs carrying premature stop codons. These findings have introduced new hopes for the development of a pharmacological approach to the ß039 thalassemia therapy. While several strategies, designed to enhance translational read-through, have been reported to inhibit NMD efficiency concomitantly, experimental tools for systematic analysis of mammalian NMD inhibition by translational read-through are lacking. RESULTS: We developed a human cellular model of the ß039 thalassemia mutation with UPF-1 suppressed and showing a partial NMD suppression. CONCLUSIONS: This novel cellular model could be used for the screening of molecules exhibiting preferential read-through activity allowing a great rescue of the mutated transcripts.

BCL11A mRNA Targeting by miR-210: A Possible Network Regulating γ-Globin Gene Expression.

The involvement of microRNAs in the control of repressors of human γ-globin gene transcription has been firmly demonstrated, as described for the miR-486-3p mediated down-regulation of BCL11A. On the other hand, we have reported that miR-210 is involved in erythroid differentiation and, possibly, in γ-globin gene up-regulation. In the present study, we have identified the coding sequence of BCL11A as a possible target of miR-210. The following results sustain this hypothesis: (a) interactions between miR-210 and the miR-210 BCL11A site were demonstrated by SPR-based biomolecular interaction analysis (BIA); (b) the miR-210 site of BCL11A is conserved through molecular evolution; (c) forced expression of miR-210 leads to decrease of BCL11A-XL and increase of γ-globin mRNA content in erythroid cells, including erythroid precursors isolated from ß-thalassemia patients. Our study suggests that the coding mRNA sequence of BCL11A can be targeted by miR-210. In addition to the theoretical point of view, these data are of interest from the applied point of view, supporting a novel strategy to inhibit BCL11A by mimicking miR-210 functions, accordingly with the concept supported by several papers and patent applications that inhibition of BCL11A is an efficient strategy for fetal hemoglobin induction in the treatment of ß-thalassemia.

An Aγ-globin G->A gene polymorphism associated with ß039 thalassemia globin gene and high fetal hemoglobin production.

BACKGROUND: Increase of the expression of γ-globin gene and high production of fetal hemoglobin (HbF) in ß-thalassemia patients is widely accepted as associated with a milder or even asymptomatic disease. The search for HbF-associated polymorphisms (such as the XmnI, BCL11A and MYB polymorphisms) has recently gained great attention, in order to stratify ß-thalassemia patients with respect to expectancy of the first transfusion, need for annual intake of blood, response to HbF inducers (the most studied of which is hydroxyurea). METHODS: Aγ-globin gene sequencing was performed on genomic DNA isolated from a total of 75 ß-thalassemia patients, including 31 ß039/ß039, 33 ß039/ß+IVSI-110, 9 ß+IVSI-110/ß+IVSI-110, one ß0IVSI-1/ß+IVSI-6 and one ß039/ß+IVSI-6. RESULTS: The results show that the rs368698783 polymorphism is present in ß-thalassemia patients in the 5'UTR sequence (+25) of the Aγ-globin gene, known to affect the LYAR (human homologue of mouse Ly-1 antibody reactive clone) binding site 5'-GGTTAT-3'. This Aγ(+25 G->A) polymorphism is associated with the Gγ-globin-XmnI polymorphism and both are linked with the ß039-globin gene, but not with the ß+IVSI-110-globin gene. In agreement with the expectation that this mutation alters the LYAR binding activity, we found that the Aγ(+25 G->A) and Gγ-globin-XmnI polymorphisms are associated with high HbF in erythroid precursor cells isolated from ß039/ß039 thalassemia patients. CONCLUSIONS: As a potential explanation of our findings, we hypothesize that in ß-thalassemia the Gγ-globin-XmnI/Aγ-globin-(G->A) genotype is frequently under genetic linkage with ß0-thalassemia mutations, but not with the ß+-thalassemia mutation here studied (i.e. ß+IVSI-110) and that this genetic combination has been selected within the population of ß0-thalassemia patients, due to functional association with high HbF. Here we describe the characterization of the rs368698783 (+25 G->A) polymorphism of the Aγ-globin gene associated in ß039 thalassemia patients with high HbF in erythroid precursor cells.

Postnatal and non-invasive prenatal detection of ß-thalassemia mutations based on Taqman genotyping assays.

The ß-thalassemias are genetic disorder caused by more than 200 mutations in the ß-globin gene, resulting in a total (ß0) or partial (ß+) deficit of the globin chain synthesis. The most frequent Mediterranean mutations for ß-thalassemia are: ß039, ß+IVSI-110, ß+IVSI-6 and ß0IVSI-1. Several molecular techniques for the detection of point mutations have been developed based on the amplification of the DNA target by polymerase chain reaction (PCR), but they could be labor-intensive and technically demanding. On the contrary, TaqMan® genotyping assays are a simple, sensitive and versatile method suitable for the single nucleotide polymorphism (SNP) genotyping affecting the human ß-globin gene. Four TaqMan® genotyping assays for the most common ß-thalassemia mutations present in the Mediterranean area were designed and validated for the genotype characterization of genomic DNA extracted from 94 subjects comprising 25 healthy donors, 33 healthy carriers and 36 ß-thalassemia patients. In addition, 15 specimens at late gestation (21-39 gestational weeks) and 11 at early gestation (5-18 gestational weeks) were collected from pregnant women, and circulating cell-free fetal DNAs were extracted and analyzed with these four genotyping assays. We developed four simple, inexpensive and versatile genotyping assays for the postnatal and prenatal identification of the thalassemia mutations ß039, ß+IVSI-110, ß+IVSI-6, ß0IVSI-1. These genotyping assays are able to detect paternally inherited point mutations in the fetus and could be efficiently employed for non-invasive prenatal diagnosis of ß-globin gene mutations, starting from the 9th gestational week.

A validated cellular biobank for ß-thalassemia.

BACKGROUND: Cellular biobanking is a key resource for collaborative networks planning to use same cells in studies aimed at solving a variety of biological and biomedical issues. This approach is of great importance in studies on ß-thalassemia, since the recruitment of patients and collection of specimens can represent a crucial and often limiting factor in the experimental planning. METHODS: Erythroid precursor cells were obtained from 72 patients, mostly ß-thalassemic, expanded and cryopreserved. Expression of globin genes was analyzed by real time RT-qPCR. Hemoglobin production was studied by HPLC. RESULTS: In this paper we describe the production and validation of a Thal-Biobank constituted by expanded erythroid precursor cells from ß-thalassemia patients. The biobanked samples were validated for maintenance of their phenotype after (a) cell isolation from same patients during independent phlebotomies, (b) freezing step in different biobanked cryovials, (c) thawing step and analysis at different time points. Reproducibility was confirmed by shipping the frozen biobanked cells to different laboratories, where the cells were thawed, cultured and analyzed using the same standardized procedures. The biobanked cells were stratified on the basis of their baseline level of fetal hemoglobin production and exposed to fetal hemoglobin inducers. CONCLUSION: The use of biobanked cells allows stratification of the patients with respect to fetal hemoglobin production and can be used for determining the response to the fetal hemoglobin inducer hydroxyurea and to gene therapy protocols with reproducible results.