Coinheritance of PIEZO1 variants and multi-locus red blood cell defects account for the symptomatic phenotype in beta-thalassemia carriers.

Workflow of the study with some examples of the achieved results.

Hb Alessandria [ß37(C3)Trp→Leu; HBB: c.113G>T]: a Novel Variant on the ß-Globin Chain with Slightly Increased Affinity for Oxygen Detected by Capillary Electrophoresis.

We report a novel mutation on the ß-globin gene in a 68-year-old woman of Sicilian origin living in Alessandria, Italy. This mutation produces a hemoglobin (Hb) variant of Hb A that was detected by the capillary electrophoresis (CE) method during measurement of Hb A1c. The variant Hb did not separate from Hb A using different high performance liquid chromatography (HPLC) instruments. Direct DNA sequencing revealed a G>T transversion at codon 37 and subsequent substitution of a tryptophan residue for a leucine residue. The new Hb variant was named Hb Alessandria [ß37(C3)Trp→Leu; HBB: c.113G>T]. The p50 value was slightly decreased while the stability test at 37 °C in isopropyl alcohol and the main erythrocyte parameters were normal. Overall, the patient appeared clinically normal.

Case Report: Clinical and Hematological Characteristics of ÎµÎ³Î´ß Thalassemia in an Italian Patient.

Introduction: ÎµÎ³Î´ß thalassemia is a rare form of ß-thalassemia mostly described in children originating from Northern Europe. Only anecdotic cases from the Mediterranean area are reported. The diagnosis is challenging, considering the rarity of the disease and its heterogeneous clinical presentation. Most patients have neonatal microcytic anemia, sometimes requiring in utero and/or neonatal transfusions, and typically improving with age. Case Description: We report on an Italian newborn presenting with severe neonatal anemia that required red blood cell transfusion. After the first months of life, hemoglobin levels improved with residual very low mean corpuscular volume. ß and α thalassemia, IRIDA syndrome, and sideroblastic anemia were excluded. Finally, a diagnosis of ÎµÎ³Î´ß thalassemia was made after microarray analysis of single nucleotide polymorphisms revealed a 26 kb single copy loss of chromosome 11p15.4, including the HBD, HBBP1, HBG1, and HBB genes. Conclusions: Despite its rarity, the diagnosis of ÎµÎ³Î´ß thalassemia should be considered in newborns with severe neonatal anemia requiring in utero and/or neonatal transfusions, but also in older infants with microcytic anemia, after excluding more prevalent red blood cell disorders.

Hemoglobin A2 and Heterogeneous Diagnostic Relevance Observed in Eight New Variants of the Delta Globin Gene.

BACKGROUND: Hemoglobin A (Hb A) (α2ß2) in the normal adult subject constitutes 96-98% of hemoglobin, and Hb F is normally less than 1%, while for hemoglobin A2 (Hb A2) (α2δ2), the normal reference values are between 2.0 and 3.3%. It is important to evaluate the presence of possible delta gene mutations in a population at high risk for globin gene defects in order to correctly diagnose the ß-thalassemia carrier. METHODS: The most used methods for the quantification of Hb A2 are based on automated high performance liquid chromatography (HPLC) or capillary electrophoresis (CE). In particular Hb analyses were performed by HPLC on three dedicated devices. DNA analyses were performed according to local standard protocols. RESULTS: Here, we described eight new δ-globin gene variants discovered and characterized in some laboratories in Northern Italy in recent years. These new variants were added to the many already known Hb A2 variants that were found with an estimated frequency of about 1-2% during the screening tests in our laboratories. CONCLUSIONS: The knowledge recognition of the delta variant on Hb analysis and accurate molecular characterization is crucial to provide an accurate definitive thalassemia diagnosis, particularly in young subjects who would like to ask for a prenatal diagnosis or preimplantation genetic diagnosis.

Hemoglobin Yamagata [ß132(H10)Lys→Asn; (HBB: c.399A>T)]: a mosaic to be put together.

OBJECTIVES: Artifactually altered glycated hemoglobin (HbA1c) concentrations are frequently linked to hemoglobin (Hb) variants. Their expression and detection require in-depth analysis. METHODS: Cation exchange high performance liquid chromatography (HPLC) (Bio-Rad Variant™ II; Trinity Biotech Premier Hb9210 Resolution), capillary electrophoresis (CE) (Sebia Capillarys 2 Flex Piercing) and mass spectrometry (MS) (Waters) were used for variant detection; Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA) and next generation sequencing (NGS) were used for DNA analysis; HbA1c was measured with cation exchange HPLC (Bio-Rad Variant™ II; Arkray Adams HA-8180V; Tosoh HLC-723 G7), CE (Sebia Capillarys 2 Flex Piercing), boronate affinity HPLC (Trinity Biotech Hb9210 Premier), immunoassay (Cobas c501 Tina-quant HbA1c Gen. 3; Nihon Kohden CHM-4100 Celltac chemi HbA1c HA-411V) and enzymatic assay (Abbott Architect c 8000 HbA1c). RESULTS: Hb Yamagata [ß132(H10)Lys→Asn; (HBB: c.399A>T)] was identified in the proband by MS after the observation of an abnormal peak in HPLC and CE. A mosaic expression of this variant was detected by NGS (mutant: 8%; wild type: 92%), after negative results in Sanger sequencing. Hb Yamagata interfered with HbA1c measurements by cation exchange HPLC and CE whereas immuno and enzymatic assay values showed good agreement with boronate affinity HPLC measurement. CONCLUSIONS: A mosaicism of Hb Yamagata was found in a patient with altered HbA1c values. This rare gene variant was detected only by advanced technologies as MS and NGS. The variant interfered with common HbA1c determination methods.

Hb A2-Pistoia [δ89(F5)Ser→Asn, HBD: c.269G>a]: a Novel Mutation on the δ-Globin Gene in an Italian Child.

We describe a new hemoglobin (Hb) variant, found in a 6-year-old Italian male living in Pistoia, Italy. An abnormal pattern compatible with a Hb A2 variant was observed on capillary electrophoresis (CE); direct sequencing revealed a transition at codon 89 of the δ gene (HBD: c.269G>A) changing serine into asparagine. The variant was also identified as Hb A2-Pistoia according to the traditional nomenclature and no other globin defect was present. The observation and description of this Hb A2 variant contributes to the number and heterogeneity of mutations of the δ-globin gene in the Mediterranean Area.

Culture Medium Supplements Derived from Human Platelet and Plasma: Cell Commitment and Proliferation Support.

Present cell culture medium supplements, in most cases based on animal sera, are not fully satisfactory especially for the in vitro expansion of cells intended for human cell therapy. This paper refers to (i) an heparin-free human platelet lysate (PL) devoid of serum or plasma components (v-PL) and (ii) an heparin-free human serum derived from plasma devoid of PL components (Pl-s) and to their use as single components or in combination in primary or cell line cultures. Human mesenchymal stem cells (MSC) primary cultures were obtained from adipose tissue, bone marrow, and umbilical cord. Human chondrocytes were obtained from articular cartilage biopsies. In general, MSC expanded in the presence of Pl-s alone showed a low or no proliferation in comparison to cells grown with the combination of Pl-s and v-PL. Confluent, growth-arrested cells, either human MSC or human articular chondrocytes, treated with v-PL resumed proliferation, whereas control cultures, not supplemented with v-PL, remained quiescent and did not proliferate. Interestingly, signal transduction pathways distinctive of proliferation were activated also in cells treated with v-PL in the absence of serum, when cell proliferation did not occur, indicating that v-PL could induce the cell re-entry in the cell cycle (cell commitment), but the presence of serum proteins was an absolute requirement for cell proliferation to happen. Indeed, Pl-s alone supported cell growth in constitutively activated cell lines (U-937, HeLa, HaCaT, and V-79) regardless of the co-presence of v-PL. Plasma- and plasma-derived serum were equally able to sustain cell proliferation although, for cells cultured in adhesion, the Pl-s was more efficient than the plasma from which it was derived. In conclusion, the cells expanded in the presence of the new additives maintained their differentiation potential and did not show alterations in their karyotype.

First Characterization of Human Amniotic Fluid Stem Cell Extracellular Vesicles as a Powerful Paracrine Tool Endowed with Regenerative Potential.

Human amniotic fluid stem cells (hAFS) have shown a distinct secretory profile and significant regenerative potential in several preclinical models of disease. Nevertheless, little is known about the detailed characterization of their secretome. Herein we show for the first time that hAFS actively release extracellular vesicles (EV) endowed with significant paracrine potential and regenerative effect. c-KIT+ hAFS were isolated from leftover samples of amniotic fluid from prenatal screening and stimulated to enhance EV release (24 hours 20% O2 versus 1% O2 preconditioning). The capacity of the c-KIT+ hAFS-derived EV (hAFS-EV) to induce proliferation, survival, immunomodulation, and angiogenesis were investigated in vitro and in vivo. The hAFS-EV regenerative potential was also assessed in a model of skeletal muscle atrophy (HSA-Cre, SmnF7/F7 mice), in which mouse AFS transplantation was previously shown to enhance muscle strength and survival. hAFS secreted EV ranged from 50 up to 1,000 nm in size. In vitro analysis defined their role as biological mediators of regenerative, paracrine effects while their modulatory role in decreasing skeletal muscle inflammation in vivo was shown for the first time. Hypoxic preconditioning significantly induced the enrichment of exosomes endowed with regenerative microRNAs within the hAFS-EV. In conclusion, this is the first study showing that c-KIT+ hAFS dynamically release EV endowed with remarkable paracrine potential, thus representing an appealing tool for future regenerative therapy. Stem Cells Translational Medicine 2017;6:1340-1355.

A humanized system to expand in vitro amniotic fluid-derived stem cells intended for clinical application.

BACKGROUND AIMS: The amniotic fluid is a new source of multipotent stem cells with therapeutic potential for human diseases. In agreement with the regulatory requirement to reduce and possibly to avoid animal-derived reagents in the culture of cells intended for cell therapy, bovine serum, the most common supplement in the culture medium, was replaced by human platelet-derived growth factors. METHODS: We tested a new culture medium to expand monolayers of human amniotic fluid stem cells (hAFSC) for clinical use. The AFSC were isolated by c-Kit selection and expanded in media supplemented with either bovine serum or a human platelet lysate (Lyset). RESULTS: We compared proliferation kinetics, colony-forming unit percentage, multilineage differentiation, immunophenotypic characterization and inhibition of peripheral blood mononuclear cell proliferation of the two AFSC cell cultures and we found no significant differences. Moreover, the karyotype analysis of the cells expanded in the presence of the platelet lysate did not present cytogenetic abnormalities and in vitro and in vivo studies revealed no cell tumorigenicity. CONCLUSIONS: Platelet derivatives represent a rich source of growth factors that can play a safety role in the homeostasis, proliferation and remodeling of tissue healing. We propose human platelet extracts as a preferential alternative to animal serum for the expansion of stem cells for clinical applications.

Clinical aspects of Fanconi anemia individuals with the same mutation of FANCF identified by next generation sequencing.

BACKGROUND: Fanconi anemia (FA) is a rare genetic disease characterized by congenital malformations, aplastic anemia and increased risk of developing malignancies. FA is genetically heterogeneous as it is caused by at least 17 different genes. Among these, FANCA, FANCC, and FANCG account for approximately 85% of the patients whereas the remaining genes are mutated in only a small percentage of cases. For this reason, the molecular diagnostic process is complex and not always extended to all the FA genes, preventing the characterization of individuals belonging to rare groups. METHODS: The FA genes were analyzed using a next generation sequencing approach in two unrelated families. RESULTS: The analysis identified the same, c.484_485del, homozygous mutation of FANCF in both families. A careful examination of three electively aborted fetuses in one family and one affected girl in the other indicated an association of the FANCF loss-of-function mutation with a severe phenotype characterized by multiple malformations. CONCLUSION: The systematic use of next generation sequencing will allow the recognition of individuals from rare complementation groups, a better definition of their clinical phenotypes, and consequently, an appropriate genetic counseling.

Characterization of a complex rearrangement involving chromosomes 1, 4 and 8 by FISH and array-CGH.

Complex chromosomal rearrangements (CCRs) are structural aberrations involving more than two chromosomes with at least three breakpoints. CCRs can be divided into familial and de novo. Balanced CCR are extremely rare in humans and are at high risk of producing unbalanced gametes. Individuals with balanced CCR are usually phenotipically normal but report fertility problems, recurrent miscarriages or congenital anomalies in newborn offsprings as consequence of either meiotic failure or imbalanced chromosomes segregation.We describe the case of an unbalanced CCR involving chromosomes 1, 4 and 8 found in a girl with developmental delay, hexadactilia and microcephaly. The rearrangement, apparently balanced at a standard karyotype analysis and of maternal origin, was demonstrated to be unbalanced by array-CGH and FISH. In conclusion our study underlines the importance of the combined use of a quantitative technique, as array-CGH, to detect criptic segmental aneuploidies, and a qualitative tool, as FISH analysis, to physically map the localization of the chromosome segments involved, in order to realize the exact nature that underlies a chromosomal rearrangement.

Recruitment of host's progenitor cells to sites of human amniotic fluid stem cells implantation.

The amniotic fluid is a new source of multipotent stem cells with a therapeutic potential for human diseases. Cultured at low cell density, human amniotic fluid stem cells (hAFSCs) were still able to generate colony-forming unit-fibroblast (CFU-F) after 60 doublings, thus confirming their staminal nature. Moreover, after extensive in vitro cell expansion hAFSCs maintained a stable karyotype. The expression of genes, such as SSEA-4, SOX2 and OCT3/4 was confirmed at early and later culture stage. Also, hAFSCs showed bright expression of mesenchymal lineage markers and immunoregulatory properties. hAFSCs, seeded onto hydroxyapatite scaffolds and subcutaneously implanted in nude mice, played a pivotal role in mounting a response resulting in the recruitment of host's progenitor cells forming tissues of mesodermal origin such as fat, muscle, fibrous tissue and immature bone. Implanted hAFSCs migrated from the scaffold to the skin overlying implant site but not to other organs. Given their in vivo: (i) recruitment of host progenitor cells, (ii) homing towards injured sites and (iii) multipotentiality in tissue repair, hAFSCs are a very appealing reserve of stem cells potentially useful for clinical application in regenerative medicine.

Multipotent mesenchymal stromal cells from amniotic fluid: solid perspectives for clinical application.

BACKGROUND: Mesenchymal stromal cells are multipotent cells considered to be of great promise for use in regenerative medicine. However, the cell dose may be a critical factor in many clinical conditions and the yield resulting from the ex vivo expansion of mesenchymal stromal cells derived from bone marrow may be insufficient. Thus, alternative sources of mesenchymal stromal cells need to be explored. In this study, mesenchymal stromal cells were successfully isolated from second trimester amniotic fluid and analyzed for chromosomal stability to validate their safety for potential utilization as a cell therapy product. DESIGN AND METHODS: Mesenchymal stromal cells were expanded up to the sixth passage starting from amniotic fluid using different culture conditions to optimize large-scale production. RESULTS: The highest number of mesenchymal stromal cells derived from amniotic fluid was reached at a low plating density; in these conditions the expansion of mesenchymal stromal cells from amniotic fluid was significantly greater than that of adult bone marrow-derived mesenchymal stromal cells. Mesenchymal stromal cells from amniotic fluid represent a relatively homogeneous population of immature cells with immunosuppressive properties and extensive proliferative potential. Despite their high proliferative capacity in culture, we did not observe any karyotypic abnormalities or transformation potential in vitro nor any tumorigenic effect in vivo. CONCLUSIONS: Fetal mesenchymal stromal cells can be extensively expanded from amniotic fluid, showing no karyotypic abnormalities or transformation potential in vitro and no tumorigenic effect in vivo. They represent a relatively homogeneous population of immature mesenchymal stromal cells with long telomeres, immunosuppressive properties and extensive proliferative potential. Our results indicate that amniotic fluid represents a rich source of mesenchymal stromal cells suitable for banking to be used when large amounts of cells are required.

Clinical scale ex vivo expansion of cord blood-derived outgrowth endothelial progenitor cells is associated with high incidence of karyotype aberrations.

OBJECTIVE: Endothelial progenitor cells (EPCs) are involved in neovessel formation. So far, therapeutic angiogenesis is hampered by the low frequency and limited proliferative potential of these cells isolated from peripheral blood. Recently, it has been shown that cord blood-derived EPCs (CB EPCs) can be ex vivo expanded on a clinical scale. In this study, we evaluated the expansion potential of CB EPCs together with their phenotypic, functional, and chromosomal stability over time. MATERIALS AND METHODS: Flow cytometry, in vitro tube formation, and proliferation assays were performed to characterize CB EPC-derived cells. Chromosomal stability was evaluated by karyotype analysis. In vitro and in vivo tumorigenicity was evaluated by soft agar assay and injection into nonobese diabetic/severe combined immunodeficient mice, respectively. RESULTS: We showed that CB EPC-derived cells displayed phenotypic and functional features of EPCs, although a process of maturation was observed over time. Although we confirmed that CB EPCs have a greater expansion potential compared to peripheral blood EPCS, we observed a high incidence of cytogenetic alterations (71%) in the expanded endothelial cell population, even at early times of culture. In two cases, spontaneous transformation in vitro was documented, but none of the samples tested showed tumorigenic potential in vivo. Conversely, no karyotype alterations have been observed on peripheral blood EPCs-derived cells. CONCLUSIONS: We confirm that CB represents a good source for clinical ex vivo expansion of EPCs. However, because of high frequency of karyotype alterations, these cells cannot be considered free of risk in clinical application.

Familial robertsonian 13;14 translocation with mental retardation and epilepsy.

Familial reports of a robertsonian translocation in more than two generations are rare. We report three generations (a daughter, the mother, and the mother's father) with a heterozygous, balanced robertsonian translocation t(13;14)(q11;q11). Central nervous system disease was present, but differentially expressed, in generations I and III. The daughter presented with mental delay and epilepsy, and the mother was apparently healthy, whereas the mother's father was again symptomatic, with borderline intelligence. Fluorescent in situ hybridization analysis was performed to exclude a loss or gain of chromosomal material. No uniparental disomy was present. We concluded that genetic counseling in the presence of this rearrangement was extremely difficult, independent of the affected parent being symptomatic or asymptomatic.

Global developmental delay, osteopenia and ectodermal defect: a new syndrome.

UNLABELLED: Global developmental delay is a serious social problem. It is often unrecognized and the phenotypes are inadequately studied. To investigate the phenotypes of children with aspecific central nervous system (CNS) impairment (poor speech, maladaptive behavioral symptoms such as temper tantrums, aggressiveness, poor concentration and attention, impulsiveness, and mental retardation). SETTING: Tertiary care hospital. PATIENTS: Three children (two male siblings, and one unrelated girl). METHODS: We used the results from clinical neurological evaluations; imaging and electrodiagnostic studies; metabolic and genetic tests; skin biopsies and bone mineral densitometry. All three children suffered from (A) global developmental delay, (B) osteopenia, and (C) identical skin defects. The skin ultrastructural abnormalities were abnormal keratin differentiation, consisting of hyperkeratosis and granular layer thickening; sweat gland abnormalities, consisting of focal, cytoplasmic clear changes in eccrine secretory cells; and melanocyte abnormalities, with both morphological changes (reduced number and size without evident dendritic processes), and functional changes (defects in the migration of melanosomes in the keratinocytes). These patients present a previously unrecognized syndrome. We retain useful to report this new association, to be recognized, in the next future, as a specific key-sign of a well-defined genetic defect.