Fyn Kinase Is Involved in EPO Receptor Signaling and Is Required to Harmonize the Response to Oxidation.

DISCLOSURES: No relevant conflicts of interest to declare.

Fyn kinase is a novel modulator of erythropoietin signaling and stress erythropoiesis.

The signaling cascade induced by the interaction of erythropoietin (EPO) with its receptor (EPO-R) is a key event of erythropoiesis. We present here data indicating that Fyn, a Src-family-kinase, participates in the EPO signaling-pathway, since Fyn-/- mice exhibit reduced Tyr-phosphorylation of EPO-R and decreased STAT5-activity. The importance of Fyn in erythropoiesis is also supported by the blunted responsiveness of Fyn-/- mice to stress erythropoiesis. Fyn-/- mouse erythroblasts adapt to reactive oxygen species (ROS) by activating the redox-related-transcription-factor Nrf2. However, since Fyn is a physiologic repressor of Nrf2, absence of Fyn resulted in persistent-activation of Nrf2 and accumulation of nonfunctional proteins. ROS-induced over-activation of Jak2-Akt-mTOR-pathway and repression of autophagy with perturbation of lysosomal-clearance were also noted. Treatment with Rapamycin, a mTOR-inhibitor and autophagy activator, ameliorates Fyn-/- mouse baseline erythropoiesis and erythropoietic response to oxidative-stress. These findings identify a novel multimodal action of Fyn in the regulation of normal and stress erythropoiesis.

The role of TMPRSS6 and HFE variants in iron deficiency anemia in celiac disease.

We investigated the role of HFE C282Y, H63D, and TMPRSS6 A736V variants in the pathogenesis of iron deficiency anemia (IDA) in celiac disease (CD) patients, at diagnosis and after 1 year of gluten-free diet (GFD). Demographic and clinical features were prospectively recorded for all CD patients between 2013 and 2017. C282Y, H63D, and A736V variants were evaluated for CD patients and controls. Finally, 505 consecutive CD patients and 539 age-matched control subjects were enrolled. At diagnosis, 229 CD subjects had IDA (45.3%), with a subgroup of anemic patients (45.4%) presented persistent IDA at follow-up. C282Y allele frequency was significantly increased in CD compared with controls (1.1% vs 0.2%, P = .001), whereas H63D and A736V allele frequencies were similar among patients and controls (P = .92 and .84, respectively). At diagnosis, C282Y variant in anemic CD patients was significantly increased compared to nonanemic group (2% and 0.5%, P = .04). At follow-up, A736V was significantly increased in IDA persistent than in IDA not persistent (57.7% vs 35.2%, P < .0001). CD patients with H63D mutation showed higher Hb, MCV, serum iron, and ferritin levels than subjects without HFE mutations. Decreased hepcidin values were observed in anemic compared to nonanemic subjects at follow-up (1.22 ± 1.14 vs 2.08 ± 2.15, P < .001). This study suggests a protective role of HFE in IDA CD patients and confirms the role of TMPRSS6 in predicting oral iron response modulating hepcidin action on iron absorption. Iron supplementation therapeutic management in CD could depend on TMPRSS6 genotype that could predict persistent IDA despite iron supplementation and GFD.

Novel compound heterozygous mutations in BCS1L gene causing Bjornstad syndrome in two siblings.

Bjornstad syndrome is a rare condition characterized by pili torti and sensorineural hearing loss associated with pathological variations in BCS1L. Mutations in this gene are also associated with the more severe complex III deficiency and GRACILE syndrome. We report the first Italian patients with Bjornstad syndrome, two siblings with pili torti and sensorineural hearing loss, in whom we detected two novel compound heterozygous mutations in BCS1L. A thorough clinical evaluation did not reveal any features consistent with complex III deficiency or GRACILE syndrome.

Iron-refractory iron deficiency anemia (IRIDA) cases with 2 novel TMPRSS6 mutations.

Iron-refractory iron deficiency anemia (IRIDA) is a rarely diagnosed autosomal recessive disorder that presents with hypochromic, microcytic anemia due to mutations in TMPRSS6, which encodes matriptase-2. Contrary to classical iron deficiency anemia, serum hepcidin levels are found to be elevated in this disorder. Here, we report 5 cases from 4 unrelated families with inadequate response to iron therapy, who were consequently diagnosed as IRIDA. The mean age of the cases at diagnosis was 5.0 years (range: 0.7-11.3 years). All cases were either homozygous or compound heterozygous for missense or frameshift mutations in the TMPRSS6 gene, 2 of the mutations being novel (Cys410Ser and Leu689Pro). IRIDA should be considered in patients with findings of iron deficiency anemia unresponsive to oral iron therapy, whose serum ferritin levels are found normal or elevated.

Multiple clinical forms of dehydrated hereditary stomatocytosis arise from mutations in PIEZO1.

Autosomal dominant dehydrated hereditary stomatocytosis (DHSt) usually presents as a compensated hemolytic anemia with macrocytosis and abnormally shaped red blood cells (RBCs). DHSt is part of a pleiotropic syndrome that may also exhibit pseudohyperkalemia and perinatal edema. We identified PIEZO1 as the disease gene for pleiotropic DHSt in a large kindred by exome sequencing analysis within the previously mapped 16q23-q24 interval. In 26 affected individuals among 7 multigenerational DHSt families with the pleiotropic syndrome, 11 heterozygous PIEZO1 missense mutations cosegregated with disease. PIEZO1 is expressed in the plasma membranes of RBCs and its messenger RNA, and protein levels increase during in vitro erythroid differentiation of CD34(+) cells. PIEZO1 is also expressed in liver and bone marrow during human and mouse development. We suggest for the first time a correlation between a PIEZO1 mutation and perinatal edema. DHSt patient red cells with the R2456H mutation exhibit increased ion-channel activity. Functional studies of PIEZO1 mutant R2488Q expressed in Xenopus oocytes demonstrated changes in ion-channel activity consistent with the altered cation content of DHSt patient red cells. Our findings provide direct evidence that R2456H and R2488Q mutations in PIEZO1 alter mechanosensitive channel regulation, leading to increased cation transport in erythroid cells.

Functional and clinical impact of novel TMPRSS6 variants in iron-refractory iron-deficiency anemia patients and genotype-phenotype studies.

Iron-refractory iron-deficiency anemia (IRIDA) is a rare autosomal-recessive disorder characterized by hypochromic microcytic anemia, low transferrin saturation, and inappropriate high levels of the iron hormone hepcidin. The disease is caused by variants in the transmembrane protease serine 6 (TMPRSS6) gene that encodes the type II serine protease matriptase-2, a negative regulator of hepcidin transcription. Sequencing analysis of the TMPRSS6 gene in 21 new IRIDA patients from 16 families with different ethnic origin reveal 17 novel mutations, including the most frequent mutation in Southern Italy (p.W590R). Eight missense mutations were analyzed in vitro. All but the p.T287N variant impair matriptase-2 autoproteotylic activation, decrease the ability to cleave membrane HJV and inhibit the HJV-dependent hepcidin activation. Genotype-phenotype studies in IRIDA patients have been so far limited due to the relatively low number of described patients. Our genotype-phenotype correlation analysis demonstrates that patients carrying two nonsense mutations present a more severe anemia and microcytosis and higher hepcidin levels than the other patients. We confirm that TMPRSS6 mutations are spread along the gene and that mechanistically they fully or partially abrogate hepcidin inhibition. Genotyping IRIDA patients help in predicting IRIDA severity and may be useful for predicting response to iron treatment.

Resveratrol accelerates erythroid maturation by activation of FoxO3 and ameliorates anemia in beta-thalassemic mice.

Resveratrol, a polyphenolic-stilbene, has received increased attention in the last decade due to its wide range of biological activities. Beta(ß)-thalassemias are inherited red cell disorders, found worldwide, characterized by ineffective erythropoiesis and red cell oxidative damage with reduced survival. We evaluated the effects of low-dose-resveratrol (5 µM) on in vitro human erythroid differentiation of CD34(+) from normal and ß-thalassemic subjects. We found that resveratrol induces accelerated erythroid-maturation, resulting in the reduction of colony-forming units of erythroid cells and increased intermediate and late erythroblasts. In sorted colony-forming units of erythroid cells resveratrol activates Forkhead-box-class-O3, decreases Akt activity and up-regulates anti-oxidant enzymes as catalase. In an in vivo murine model for ß-thalassemia, resveratrol (2.4 mg/kg) reduces ineffective erythropoiesis, increases hemoglobin levels, reduces reticulocyte count and ameliorates red cell survival. In both wild-type and ß-thalassemic mice, resveratrol up-regulates scavenging enzymes such as catalase and peroxiredoxin-2 through Forkhead-box-class-O3 activation. These data indicate that resveratrol inhibits Akt resulting in FoxO3 activation with upregulation of cytoprotective systems enabling the pathological erythroid precursors to resist the oxidative damage and continue to differentiate. Our data suggest that the dual effect of resveratrol on erythropoiesis through activation of FoxO3 transcriptional factor combined with the amelioration of oxidative stress in circulating red cells may be considered as a potential novel therapeutic strategy in treating ß-thalassemia.

Human skin-derived keratinocytes and fibroblasts co-cultured on 3D poly ε-caprolactone scaffold support in vitro HSC differentiation into T-lineage committed cells.

In humans, the thymus is the primary lymphoid organ able to support the development of T cells through its three-dimensional (3D) organization of the thymic stromal cells. Since a remarkable number of similarities are shared between the thymic epithelial cells (TECs) and skin-derived keratinocytes and fibroblasts, in this study we used human keratinocytes seeded with fibroblasts on the 3D poly ε-caprolactone scaffold to evaluate their ability to replace TECs in supporting T-cell differentiation from human haematopoietic stem cells (HSCs). We observed that in the multicellular biocomposite, early thymocytes expressing CD7(+)CD1a(+), peculiar markers of an initial T-cell commitment, were de novo generated. Molecular studies of genes selectively expressed during T-cell development revealed that TAL1 was down-regulated and Spi-B was up-regulated in the cell suspension, consistently with a T-cell lineage commitment. Moreover, PTCRA and RAG2 expression was detected, indicative of a recombinant activity, required for the generation of a T-cell receptor repertoire. Our results indicate that in the multicellular biocomposite, containing skin-derived elements in the absence of thymic stroma, HSCs do start differentiating toward a T-cell lineage commitment. In conclusion, the construct described in this study exerts some properties of a lymphoid organoid, suitable for future clinical applications in cell-based therapies.

Trisomy 21 with Maternally Inherited Balanced Translocation (15q;22q) in a Female Fetus: A Rare Case of Probable Interchromosomal Effect.

Chromosomal rearrangements can interfere with the disjunction and segregation of other chromosome pairs not involved in the rearrangements, promoting the occurrence of numerical abnormalities in resulting gametes and predisposition to trisomy in offspring. This phenomenon of interference is known as the interchromosomal effect (ICE). Here we report a prenatal case potentially generated by ICE. The first-trimester screening ultrasound of the pregnant woman was normal, but the NIPT indicated a high risk for three copies of chromosome 21, thus suspecting trisomy 21 (T21). After a comprehensive clinical evaluation and genetic counseling, the couple decided to undergo amniocentesis. The prenatal karyotype confirmed T21 but also showed a balanced translocation between the long arm of chromosome 15 (q22) and the long arm of chromosome 22. The parents' karyotypes also showed that the mother had the 15;22 translocation. We reviewed T21 screening methods, and we performed a literature review on ICE, a generally overlooked phenomenon. We observed that ours is the first report of a prenatal case potentially due to ICE derived from the mother. The recurrence risk of aneuploidy in the offspring of translocated individuals is likely slightly increased, but it is not possible to estimate to what extent. In addition to supporting observations, there are still open questions such as, how frequent is ICE? How much is the aneuploidy risk altered by ICE?

Iron refractory iron deficiency anemia.

Iron refractory iron deficiency anemia is a hereditary recessive anemia due to a defect in the TMPRSS6 gene encoding Matriptase-2. This protein is a transmembrane serine protease that plays an essential role in down-regulating hepcidin, the key regulator of iron homeostasis. Hallmarks of this disease are microcytic hypochromic anemia, low transferrin saturation and normal/high serum hepcidin values. The anemia appears in the post-natal period, although in some cases it is only diagnosed in adulthood. The disease is refractory to oral iron treatment but shows a slow response to intravenous iron injections and partial correction of the anemia. To date, 40 different Matriptase-2 mutations have been reported, affecting all the functional domains of the large ectodomain of the protein. In vitro experiments on transfected cells suggest that Matriptase-2 cleaves Hemojuvelin, a major regulator of hepcidin expression and that this function is altered in this genetic form of anemia. In contrast to the low/undetectable hepcidin levels observed in acquired iron deficiency, in patients with Matriptase-2 deficiency, serum hepcidin is inappropriately high for the low iron status and accounts for the absent/delayed response to oral iron treatment. A challenge for the clinicians and pediatricians is the recognition of the disorder among iron deficiency and other microcytic anemias commonly found in pediatric patients. The current treatment of iron refractory iron deficiency anemia is based on parenteral iron administration; in the future, manipulation of the hepcidin pathway with the aim of suppressing it might become an alternative therapeutic approach.

Clinical Experience with Genome-Wide Noninvasive Prenatal Screening in a Large Cohort of Twin Pregnancies.

Non-invasive prenatal screening (NIPS) in twin gestations has been shown to have high detection rates and low false-positive rates for trisomy 21, as seen in singleton pregnancies, although there have been few large cohort twin studies, genome-wide studies in particular, to date. In this study, we looked at the performance of genome-wide NIPT in a large cohort consisting of 1244 twin pregnancy samples collected over a two-year period in a single laboratory in Italy. All samples underwent an NIPS for common trisomies, with 61.5% of study participants choosing to undergo genome-wide NIPS for additional fetal anomalies (namely, rare autosomal aneuploidies and CNVs). There were nine initial no-call results, all of which were resolved upon retest. Based on our NIPS results, 17 samples were at high risk for trisomy 21, one for trisomy 18, six for a rare autosomal aneuploidy, and four for a CNV. Clinical follow-up was available for 27 out of 29 high-risk cases; a sensitivity of 100%, a specificity of 99.9%, and a PPV of 94.4% were noted for trisomy 21. Clinical follow-up was also available for 1110 (96.6%) of the low-risk cases, all of which were true negatives. In conclusion, we found that NIPS was a reliable screening approach for trisomy 21 in twin pregnancies.

A Case Report of a Feto-Placental Mosaicism Involving a Segmental Aneuploidy: A Challenge for Genome Wide Screening by Non-Invasive Prenatal Testing of Cell-Free DNA in Maternal Plasma.

Non-invasive prenatal testing (NIPT) using cell-free DNA can detect fetal chromosomal anomalies with high clinical sensitivity and specificity. In approximately 0.1% of clinical cases, the NIPT result and a subsequent diagnostic karyotype are discordant. Here we report a case of a 32-year-old pregnant patient with a 44.1 Mb duplication on the short arm of chromosome 4 detected by NIPT at 12 weeks' gestation. Amniocentesis was carried out at 18 weeks' gestation, followed by conventional and molecular cytogenetic analysis on cells from the amniotic fluid. SNP array analysis found a de novo deletion of 1.2 Mb at chromosome 4, and this deletion was found to be near the critical region of the Wolf-Hirschhorn syndrome. A normal 46,XY karyotype was identified by G-banding analysis. The patient underwent an elective termination and molecular investigations on tissues from the fetus, and the placenta confirmed the presence of type VI true fetal mosaicism. It is important that a patient receives counselling following a high-risk call on NIPT, with appropriate diagnostic analysis advised before any decisions regarding the pregnancy are taken. This case highlights the importance of genetic counselling following a high-risk call on NIPT, especially in light of the increasing capabilities of NIPT detection of sub-chromosomal deletions and duplications.

Non-Invasive Prenatal Screening: The First Report of Pentasomy X Detected by Plasma Cell-Free DNA and Karyotype Analysis.

Pentasomy X is a sex chromosome anomaly caused by the presence of three extra X chromosomes in females (49,XXXXX instead of 46,XX) and is probably due to a nondisjunction during the meiosis. So far, only five cases prenatally diagnosed were described. The main features in 49,XXXXX karyotype include severe intellectual disability with delayed speech development, short stature, facial dysmorphisms, osseous and articular abnormalities, congenital heart malformations, and skeletal and limb abnormalities. Prenatal diagnosis is often difficult due to the lack of a clear echographic sign like nuchal translucency (NT), and mostly cases were postnatally described. We report the first case of a 49,XXXXX female that was detected by non-invasive prenatal screening (NIPS), quantitative fluorescence polymerase chain reaction (QF-PCR) and a fetal karyotype.

Oxidative stress modulates heme synthesis and induces peroxiredoxin-2 as a novel cytoprotective response in ß-thalassemic erythropoiesis.

BACKGROUND: ß-thalassemic syndromes are inherited red cell disorders characterized by severe ineffective erythropoiesis and increased levels of reactive oxygen species whose contribution to ß-thalassemic anemia is only partially understood. DESIGN AND METHODS: We studied erythroid precursors from normal and ß-thalassemic peripheral CD34(+) cells in two-phase liquid culture by proteomic, reverse transcriptase polymerase chain reaction and immunoblot analyses. We measured intracellular reactive oxygen species, heme levels and the activity of δ-aminolevulinate-synthase-2. We exposed normal cells and K562 cells with silenced peroxiredoxin-2 to H(2)O(2) and generated a recombinant peroxiredoxin-2 for kinetic measurements in the presence of H(2)O(2) or hemin. RESULTS: In ß-thalassemia the increased production of reactive oxygen species was associated with down-regulation of heme oxygenase-1 and biliverdin reductase and up-regulation of peroxiredoxin-2. In agreement with these observations in ß-thalassemic cells we found decreased heme levels related to significantly reduced activity of the first enzyme of the heme pathway, δ-aminolevulinate synthase-2 without differences in its expression. We demonstrated that the activity of recombinant δ-aminolevulinate synthase-2 is inhibited by both reactive oxygen species and hemin as a protective mechanism in ß-thalassemic cells. We then addressed the question of the protective role of peroxiredoxin-2 in erythropoiesis by exposing normal cells to oxidative stress and silencing peroxiredoxin-2 in human erythroleukemia K562 cells. We found that peroxiredoxin-2 expression is up-regulated in response to oxidative stress and required for K562 cells to survive oxidative stress. We then showed that peroxiredoxin-2 binds heme in erythroid precursors with high affinity, suggesting a possible multifunctional cytoprotective role of peroxiredoxin-2 in ß-thalassemia. CONCLUSIONS: In ß-thalassemic erythroid cells the reduction of δ-aminolevulinate synthase-2 activity and the increased expression of peroxiredoxin-2 might represent two novel stress-response protective systems.

MED12 Mutation in Two Families with X-Linked Ohdo Syndrome.

X-linked intellectual deficiency (XLID) is a widely heterogeneous group of genetic disorders that involves more than 100 genes. The mediator of RNA polymerase II subunit 12 (MED12) is involved in the regulation of the majority of RNA polymerase II-dependent genes and has been shown to cause several forms of XLID, including Opitz-Kaveggia syndrome also known as FG syndrome (MIM #305450), Lujan-Fryns syndrome (MIM #309520) and the X-linked Ohdo syndrome (MIM #300895). Here, we report on two first cousins with X-linked Ohdo syndrome with a missense mutation in MED12 gene, identified through whole exome sequencing. The probands had facial features typical of X-linked Ohdo syndrome, including blepharophimosis, ptosis, a round face with a characteristic nose and a narrow mouth. Nextera DNA Exome kit (Illumina Inc., San Diego, CA, USA) was used for exome capture. The variant identified was a c.887G > A substitution in exon 7 of the MED12 gene leading to the substitution of a glutamine for a highly conserved arginine (p. Arg296Gln). Although the variant described has been previously reported in the literature, our study contributes to the expanding phenotypic spectrum of MED12-related disorders and above all, it demonstrates the phenotypic variability among different affected patients despite harboring identical mutations.

Performance of cell-free DNA sequencing-based non-invasive prenatal testing: experience on 36,456 singleton and multiple pregnancies.

BACKGROUND: This paper describes the clinical practice and performance of cell-free DNA sequencing-based non-invasive prenatal testing (NIPT) as a screening method for fetal trisomy 21, 18, and 13 (T21, T18, and T13) and sex chromosome aneuploidies (SCA) in a general Italian pregnancy population. METHODS: The AMES-accredited laboratory offers NIPT in maternal blood as a screening test for fetal T21, T18, T13 and SCA. Samples were sequenced on a NextSeq 550 (Illumina) using the VeriSeq NIPT Solution v1 assay. RESULTS: A retrospective analysis was performed on 36,456 consecutive maternal blood samples, including 35,650 singleton pregnancies, 800 twin pregnancies, and 6 triplet pregnancies. Samples were tested between April 2017 and September 2019. The cohort included 46% elevated-risk and 54% low-risk patients. A result indicative of a classic trisomy was found in 356 (1%) of singleton or twin samples: 254 T21, 69 T18, and 33 T13. In addition, 145 results (0.4%) were indicative of a SCA. Of the combined 501 screen-positive cases, 484 had confirmatory diagnostic testing. NIPT results were confirmed in 99.2% (247/249) of T21 cases, 91.2% (62/68) of T18 cases, 84.4% (27/32) of T13 cases, and 86.7% (117/135) of SCA cases. In the 35,955 cases reported as unaffected by a classic trisomy or SCA, no false negative cases were reported. Assuming that false negative results would be reported, the sensitivity of NIPT was 100.00% for T21 (95% Cl 98.47-100.0), T18 (95% Cl 94.17-100.0), and T13 (95% Cl 87.54-100.0). The specificities were 99.99% (95% Cl 99.98-100.0), 99.98% (95% Cl 99.96-100.0), 99.99% (95% Cl 99.97-100.0), and 99.95% (95% Cl 99.92-99.97) for T21, T18, T13, and SCA, respectively. CONCLUSION: This retrospective analysis of a large cohort of consecutive patients who had whole-genome sequencing-based NIPT for classic trisomies and SCA shows excellent detection rates and low false positive rates.