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Congenital dyserythropoietic anemia type I (CDAI) is an autosomal recessive inherited haematological disorder associated with moderate-to-severe anemia characterized by ineffective erythropoiesis with distinct morphological abnormalities in erythroid precursors. We present two case of congenital dyserythropoietic anemia type I in two Sicilian patients heterozygous for β 39 globin gene cod 39 C > T with marked bone marrow abnormalities, responding to treatment with alpha interferon. The diagnosis was established using routine haematological and biochemical test, light and electron microscopy; molecular analysis of the CDAN1 gene associated to the CDAI disease was performed. The response to the treatment was monitored using the hemoglobin levels, the red cell count, the reticulocyte count and the transfusional requirement. This report points out the usefulness of the treatment with interferon alpha in two Sicilian beta thalassemia carriers, in which the therapy was well tolerated without producing any side effects; in these patients the transfusion requirements after the initiation of interferon therapy decreased. [ABSTRACT FROM AUTHOR]

Objective: Thalassaemia syndromes are the most common forms of genetic disease in Sicily. The average frequency of the β-thalassaemia trait is about 6%, with a heterogeneous distribution ranging from 10% in south-eastern Sicily to 3-4% in the north or west of the island. From these data, it can be calculated that one in 270 couples is at risk for transmitting β-thalassaemia, and with an annual birth rate of 66,000, 45 new Cooley's anaemia sufferers will be born each year. Prior to starting this programme of preimplantation genetic diagnosis (PGD) for β-thalassaemia and sickle cell anaemia in Sicily, a survey was carried out to test the willingness of high-risk Sicilian couples to undergo PGD. It was found that 44.4% of the couples coming for their first prenatal diagnosis (PND), 47.1% of the couples coming for their second or further PND without previous experience of therapeutic abortion, and 72.0% of the couples undergoing PND with previous experience of therapeutic abortion were willing to undergo PGD for β-thalassaemia and sickle cell anaemia. This report concerns two cycles of clinical PGD application for β-thalassaemia with two fertile carrier couples, one with previous experiences of therapeutic abortion for an affected fetus and one with ethical problems. Materials/Methods: In both couples, both patients were heterozygous for the Cd 39 mutation. In the IVF programme, ovarian stimulation was carried out by the administration of gonadotrophin-releasing hormone analogue. Clinical PGD for β-thalassaemia was performed by polar body (PB) analysis. The first PB was removed from each oocyte using a pipette with an inner diameter of 15-30 µm. The PB was transferred to a microcentrifuge tube containing lysis buffer with proteinase K and stored at -20°C. The oocyte was micro-injected with partner's spermatozoa by standard intracytoplasmic sperm injection (ICSI) after the first PB had been removed. The morning following the ICSI procedure the second PB was removed from the oocyte. This was treated in the same way as the first PB. All tubes, containing first and second PB, were incubated at 45°C for 15 min in a thermal cycler: proteinase K was inactivated at 96°C for 20 min. The first round of polymerase chain reaction (PCR) was performed with mix 1, containing outside primers for the β-globin gene and for two highly polymorphic markers (a short tandem repeat (STR) 5' to the β-globin gene and HUMTH01). The second round reactions (mix 2) contained singular pairs of specific nested primers for the β-globin gene and polymorphic markers (STR 5' β-lobin gene and HUMTH01). A 2 µl aliquot of PCR mix 1 was added to 48 µl of PCR mix 2 and the PCR was run. The β-globin gene in all patients contained the Cd 39 (C>T) mutation. All nested PCR samples were digested with Mae I restriction enzyme and analysed on 6% polyacrylamide gels. Results/Conclusions: Of seven oocytes analysed for the first couple, four mutation-free zygotes were detected, of which two resulting embryos were transferred back to the patient. For the second couple, six oocytes were analysed: two failed to amplify in first PB reaction, one failed in the second PB reaction, and two β-thalassaemia mutation-free zygotes resulted. Two embryos resulting from these oocytes were transferred. All ethical aspects were discussed with the at-risk couples before. during and after PGD, demonstrating the high level of acceptance of the procedure. [ABSTRACT FROM AUTHOR]

Celocentesis is a new sampling tool for prenatal diagnosis available from 7 weeks in case of couples at risk for genetic diseases. In this study, we reported the feasibility of earlier prenatal diagnosis by celocentesis in four cases of cystic fibrosis and one case of cystic fibrosis and β-thalassemia co-inherited in the same fetus. Celomic fluids were aspired from the celomic cavity between 8 +2 and 9 +3 weeks of gestation and fetal cells were picked up by micromanipulator. Maternal DNA contamination was tested and target regions of fetal DNA containing parental pathogenetic variants of CFTR and HBB genes were amplified and sequenced. Four of the five fetuses resulted as being affected by cystic fibrosis and, in all cases, the women decided to interrupt the pregnancy. In the other case, the fetus presented a healthy carrier of cystic fibrosis. The results were confirmed in three cases on placental tissue. In one case, no abortive tissue was obtained. In the last case, the woman refused the prenatal diagnosis to confirm the celocentesis data; the pregnancy is ongoing without complications. This procedure provides prenatal diagnosis of monogenic diseases at least four weeks earlier than traditional procedures, reducing the anxiety of patients and providing the option for medical termination of the affected fetus at 8-10 weeks of gestation, which is less traumatic and safer than surgical termination in the second trimester.

Background and Objectives . Retinitis pigmentosa (RP) is the most common inherited rod-cone dystrophy (RCD), resulting in nyctalopia, progressive visual field, and visual acuity decay in the late stages. The autosomal dominant form (ADRP) accounts for about 20% of RPs. Among the over 30 genes found to date related to ADRP, RP1 pathogenic variants have been identified in 5-10% of cases. In a cohort of RCD patients from the Palermo province on the island of Sicily, we identified a prevalent nonsense variant in RP1 , which was associated with ADRP. The objective of our study was to analyse the clinical and molecular data of this patient cohort and to evaluate the potential presence of a founder effect. Materials and Methods . From 2005 to January 2023, 84 probands originating from Western Sicily (Italy) with a diagnosis of RCD or RP and their relatives underwent deep phenotyping, which was performed in various Italian clinical institutions. Molecular characterisation of patients and familial segregation of pathogenic variants were carried out in different laboratories using Sanger and/or next-generation sequencing (NGS). Results. Among 84 probands with RCD/RP, we found 28 heterozygotes for the RP1 variant c.2219C>G, p.Ser740* ((NM_006269.2)*, which was therefore significantly prevalent in this patient cohort. After a careful interview process, we ascertained that some of these patients shared the same pedigree. Therefore, we were ultimately able to define 20 independent family groups with no traceable consanguinity. Lastly, analysis of clinical data showed, in our patients, that the p.Ser740* nonsense variant was often associated with a late-onset and relatively mild phenotype. Conclusions . The high prevalence of the p.Ser740* variant in ADRP patients from Western Sicily suggests the presence of a founder effect, which has useful implications for the molecular diagnosis of RCD in patients coming from this Italian region. This variant can be primarily searched for in RP-affected subjects displaying compatible modes of transmission and phenotypes, with an advantage in terms of the required costs and time for analysis. Moreover, given its high prevalence, the RP1 p.Ser740* variant could represent a potential candidate for the development of therapeutic strategies based on gene editing or translational read-through therapy for suppression of nonsense variants.

The hemoglobinopathies, as β-thalassemia (β-thal) and sickle cell disease, are the most common hereditary hemolytic anemias. The increase of fetal hemoglobin (Hb F) levels can ameliorate the symptoms of hemoglobinopathies. There are several transcription factors such as MYB and SOX-6, which are involved in the regulation of Hb F. There are not enough studies investigating the association between single nucleotide polymorphisms (SNPs) of the SOX-6 and MYB genes and the variation of Hb F levels in patients affected by sickle cell disease and β-thal. We therefore decided to analyze the role of four missense variants of MYB and SOX-6 genes in the regulation of Hb F levels. In order to do so, we examinated 30 Sicilian patients affected by sickle cell disease and β-thal, to understand if these variants could also have an influence in our populations. Comparing two groups of patients with low and high levels of Hb F, we found no significant differences in the genetic distribution and allelic frequency of MYB and SOX-6 gene polymorphisms. We also created and compared a 'high producer' and 'low producer' genotype with different genes achieving the same result of no significant difference. Our results may be due either to the fact that the association between these genes and the regulation of Hb F levels are influenced by environmental history and population genetics, or to the small number of samples being analyzed.

Increased expression of fetal hemoglobin (HbF) may ameliorate the clinical course of hemoglobinopathies. Hydroxyurea (HU) is the only inducer approved for the treatment of these diseases able to stimulate HbF production but patients' response is highly variable indicating the utility of the identification of pharmacogenomic biomarkers in order to predict pharmacological treatment efficacy. To date few studies to evaluate the role of genetic determinants in HU response have been conducted showing contradictory results. In this study we analyzed BCL11A, GATA-1, KLF-1 genes and γ-globin promoter in 60 alleles from 30 hemoglobinopathies patients under HU treatment to assess the role of these markers in HU response. We did not find any association between these genetic determinants and HU response. Before treatment started, the same patients were analyzed in vitro using liquid erythroid cultures in a test able to predict their response to HU. The results of our analysis confirm the absence of pharmacogenomic biomarker associated to HU response indicating that, the quantification of γ-globin mRNA fold increase remains the only method able to predict in vivo patients response to the drug., Competing Interests: he authors declare no potential conflict of interest.

Here we review the mechanisms that determine projection neuron identity during cortical development. Pyramidal neurons in the mammalian cerebral cortex can be classified into two major classes: corticocortical projection neurons, which are concentrated in the upper layers of the cortex, and subcortical projection neurons, which are found in the deep layers. Early progenitor cells in the ventricular zone produce deep layer neurons that express transcription factors including Sox5, Fezf2, and Ctip2, which play important roles in the specification of subcortically projecting axons. Upper layer neurons are produced from progenitors in the subventricular zone, and the expression of Satb2 in these differentiating neurons is required for the formation of axonal projections that connect the two cerebral hemispheres. The Fezf2/Ctip2 and Satb2 pathways appear to be mutually repressive, thus ensuring that individual neurons adopt either a subcortical or callosal projection neuron identity at early times during development. The molecular mechanisms by which Satb2 regulates gene expression involves long-term epigenetic changes in chromatin configuration, which may enable cell fate decisions to be maintained during development.

The transcription factor nuclear factor (NF)-kappaB has been suggested to be a key mediator of the development of lymph nodes and Peyer's patches. However, targeted deletion of NF-kappaB/ Rel family members has not yet corroborated such a function. Here we report that when mice lacking the RelA subunit of NF-kappaB are brought to term by breeding onto a tumor necrosis factor receptor (TNFR)1-deficient background, the mice that are born lack lymph nodes, Peyer's patches, and an organized splenic microarchitecture, and have a profound defect in T cell-dependent antigen responses. Analyses of TNFR1/RelA-deficient embryonic tissues and of radiation chimeras suggest that the dependence on RelA is manifest not in hematopoietic cells but rather in radioresistant stromal cells needed for the development of secondary lymphoid organs.

NF-kappaB binding sites are present in the promoter regions of many acute phase and inflammatory response genes, suggesting that NF-kappaB plays an important role in the initiation of innate immune responses. However, targeted mutations of the various NF-kappaB family members have yet to identify members responsible for this critical role. RelA-deficient mice die on embryonic day 15 from TNF-alpha-induced liver degeneration. To investigate the importance of RelA in innate immunity, we genetically suppressed this embryonic lethality by breeding the RelA deficiency onto a TNFR type 1 (TNFR1)-deficient background. TNFR1/RelA-deficient mice were born healthy, but were susceptible to bacterial infections and bacteremia and died within a few weeks after birth. Hemopoiesis was intact in TNFR1/RelA-deficient newborns, but neutrophil emigration to alveoli during LPS-induced pneumonia was severely reduced relative to that in wild-type or TNFR1-deficient mice. In contrast, radiation chimeras reconstituted with RelA or TNFR1/RelA-deficient hemopoietic cells were healthy and demonstrated no defect in neutrophil emigration during LPS-induced pneumonia. Analysis of RNA harvested from the lungs of mice 4 h after LPS insufflation revealed that the induction of several genes important for neutrophil recruitment to the lung was significantly reduced in TNFR1/RelA-deficient mice relative to that in wild-type or TNFR1-deficient mice. These results suggest that TNFR1-independent activation of RelA is essential in cells of nonhemopoietic origin during the initiation of an innate immune response.

Three intragenic microsatellites of the CFTR gene, a TA and a CA repeats, namely IVS17bTA and IVS17bCA, located in intron 17b and a CA repeat (IVS8CA) located in intron 8 of the CFTR gene, were analyzed in a large sample of Italian cystic fibrosis (CF) and normal chromosomes. Linkage disequilibrium was evaluated between each marker and difference CF mutations on a total of 377 CF and 358 normal chromosomes. Our results are consistent with the hypothesis that all delta F508 chromosomes derive from a single mutational event. The same hypothesis is valid for mutations G542X, N1303K, 1717-1G-->A, which might have been originated more recently than delta F508.

Notch locus EGF-like element mutations spl, altering eye development, and AxE2, affecting wing and sensilla development, are modified by mutations at Delta. It is shown that two allele-specific suppressors of spl involve single amino acid substitutions in the 4th (Dlsup5) and 9th (Dlsup4) EGF-like elements of the Delta protein. Cultured cells producing spl or AxE2 aggregate with cells producing wild-type Delta or Dlsup5 protein, and Dlsup5-producing cells adhere to cells producing wild-type Notch protein. However, spl,AxE2, and Dlsup5 are each defective in promoting these cell affinities, as none of the mutant proteins can compete with the corresponding wild-type proteins for formation of cell aggregates. Thus, widely separated EGF-like elements of Notch and Delta appear to participate in functional molecular interactions between the proteins. Dlsup5 does not improve adhesiveness of spl in vitro, so suppression in vivo may involve altered developmental signaling by spl-Dlsup5 complexes, rather than modified cell adhesion.

Prenatal diagnosis of haemoglobin disorders is accepted to be a useful procedure to avoid births of infants with homozygous diseases. Advances in sampling and molecular techniques, such as polymerase chain reaction (PCR) and chorionic villus sampling (CVS), have made earlier and safer first-trimester prenatal diagnosis possible. However, these procedures need previous studies of at-risk couples, which can be very time-consuming when a number of different beta-thalassaemia mutations occur in the region. We describe the possibility of making a first-trimester prenatal diagnosis by cordocentesis and fetal blood analysis at the 12th week of gestation. We found no statistically significant difference (p greater than 0.05) between beta/gamma values in fetuses at the 12th and 18th weeks of gestation. In seven affected fetuses aborted at the 12th week of gestation, the diagnosis was confirmed in all cases by PCR analysis. These findings suggest that early cordocentesis could be an alternative procedure to CVS and PCR analysis.

The neurogenic genes of Drosophila have long been known to regulate cell fate decisions in the developing ectoderm. In this paper we show that these genes also control mesoderm development. Embryonic cells that express the muscle-specific gene nautilus are overproduced in each of seven neurogenic mutants (Notch, Delta, Enhancer of split, big brain, mastermind, neuralized, and almondex), at the apparent expense of neighboring, nonexpressing mesodermal cells. The mesodermal defect does not appear to be a simple consequence of associated neural hypertrophy, suggesting that the neurogenic genes may function similarly and independently in establishing cell fates in both ectoderm and mesoderm. Altered patterns of beta 3-tubulin and myosin heavy chain gene expression in the mutants indicate a role for the neurogenic genes in development of most visceral and somatic muscles. We propose that the signal produced by the neurogenic genes is a general one, effective in both ectoderm and mesoderm.

The authors report 8 diagnostic cordocentesis performed at the end of the first trimester. The indication was thalassemia (5 cases) and karyotyping (3 cases). The technique requires that the operator holds both the probe and the needle (25 G X 90 mm); the fetal blood sample ranged between 0.25 and 0.35 cc, sufficient in all cases for the diagnosis. 1 pregnancy was terminated on the basis of the diagnostic result; no complications reported at a 3-weeks follow-up in the remaining 7 patients. The first trimester cordocentesis offers several advantages if compared to CVS, especially for thalassemia prenatal diagnosis; furthermore it opens new perspectives for intrauterine transplantations. More experience is required to assess the safety of the procedure.