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Genome-Wide Association Studies (GWASs) have identified a huge number of variants associated with different traits. However, their validation through in vitro and in vivo studies often lags well behind their identification. For variants associated with traits or diseases of biomedical interest, this gap delays the development of possible therapies. This issue also impacts beta-hemoglobinopathies, such as beta-thalassemia and sickle cell disease (SCD). The definitive cures for these diseases are currently bone marrow transplantation and gene therapy. However, limitations regarding their effective use restrict their worldwide application. Great efforts have been made to identify whether modulators of fetal hemoglobin (HbF) and, to a lesser extent, hemoglobin A2 (HbA 2 ) are possible therapeutic targets. Herein, we performed the post-GWAS in vivo validation of two genes, cyclin D3 ( CCND3 ) and nuclear factor I X ( NFIX ), previously associated with HbF and HbA 2 levels. The absence of Ccnd3 expression in vivo significantly increased g (HbF) and d (HbA 2 ) globin gene expression. Our data suggest that CCND3 is a possible therapeutic target in sickle cell disease. We also confirmed the association of Nfix with γ-globin gene expression and present data suggesting a possible role for Nfix in regulating Kruppel-like transcription factor 1 ( Klf1 ), a master regulator of hemoglobin switching. This study contributes to filling the gap between GWAS variant identification and target validation for beta-hemoglobinopathies.

Sickle cell disease (SCD) is a widespread genetic disease associated with severe disability and multi-organ damage, resulting in a reduced life expectancy. None of the existing clinical treatments provide a solution for all patients. Gene therapy and fetal haemoglobin (HbF) reactivation through genetic approaches have obtained promising, but early, results in patients. Furthermore, the search for active molecules to increase HbF is still ongoing. The delta-globin gene produces the delta-globin of haemoglobin A2 (HbA2). Although expressed at a low level, HbA2 is fully functional and could be a valid anti-sickling agent in SCD. To evaluate the therapeutic potential of a strategy aimed to over-express the delta-globin gene in vivo, we crossed transgenic mice carrying a single copy of the delta-globin gene, genetically modified to be expressed at a higher level (activated), with a humanised mouse model of SCD. The activated delta-globin gene gives rise to a consistent production of HbA2, effectively improving the SCD phenotype. For the first time in vivo, these results demonstrate the therapeutic potential of delta-globin, which could lead to novel approaches to the cure of SCD., (© 2021 British Society for Haematology and John Wiley & Sons Ltd.)

Beta hemoglobinopathies are widely spread monogenic lethal diseases. Delta-globin gene activation has been proposed as a possible approach for curing these pathologies. The therapeutic potential of delta-globin, the non-alpha component of Hemoglobin A 2 (α2δ2; HbA2), has been demonstrated in a mouse model of beta thalassemia, while its anti-sickling effect, comparable to that of gamma globin, was established some time ago. Here we show that the delta-globin mRNA level is considerably increased in a Deoxyribonuclease II-alpha knockout mouse model in which type 1 interferon (interferon beta, IFNb) is activated. IFNb activation in the fetal liver improves the delta-globin mRNA level, while the beta-globin mRNA level is significantly reduced. In addition, we show that HbA2 is significantly increased in patients with multiple sclerosis under type 1 interferon treatment. Our results represent a proof of principle that delta-globin expression can be enhanced through the use of molecules. This observation is potentially interesting in view of a pharmacological approach able to increase the HbA2 level., (Copyright © 2020 Manchinu, Simbula, Caria, Musu, Perseu, Porcu, Steri, Poddie, Frau, Cocco, Manunza, Barella and Ristaldi.)

β-thalassemia and sickle cell disease are widespread fatal genetic diseases. None of the existing clinical treatments provides a solution for all patients. Two main strategies for treatment are currently being investigated: (i) gene transfer of a normal β-globin gene; (ii) reactivation of the endogenous γ-globin gene. To date, neither approach has led to a satisfactory, commonly accepted standard of care. The δ-globin gene produces the δ-globin of hemoglobin A2. Although expressed at a low level, hemoglobin A2 is fully functional and could be a valid substitute of hemoglobin A in β-thalassemia, as well as an anti-sickling agent in sickle cell disease. Previous in vitro results suggested the feasibility of transcriptional activation of the human δ-globin gene promoter by inserting a Kruppel-like factor 1 binding site. We evaluated the activation of the Kruppel-like factor 1 containing δ-globin gene in vivo in transgenic mice. To evaluate the therapeutic potential we crossed the transgenic mice carrying a single copy activated δ-globin gene with a mouse model of β-thalassemia intermedia. We show that the human δ-globin gene can be activated in vivo in a stage- and tissue-specific fashion simply by the insertion of a Kruppel-like factor 1 binding site into the promoter. In addition the activated δ-globin gene gives rise to a robust increase of the hemoglobin level in β-thalassemic mice, effectively improving the thalassemia phenotype. These results demonstrate, for the first time, the therapeutic potential of the δ-globin gene for treating severe hemoglobin disorders which could lead to novel approaches, not involving gene addition or reactivation, to the cure of β-hemoglobinopathies.

Although several genes are implicated in the pathogenesis of schizophrenia, in animal models for such a severe mental illness only some aspects of the pathology can be represented (endophenotypes). Genetically modified mice are currently being used to obtain or characterize such endophenotypes. Since its cloning and characterization CB1 receptor has increasingly become of significant physiological, pharmacological and clinical interest. Recently, its involvement in schizophrenia has been reported. Among the different approaches employed, gene targeting permits to study the multiple roles of the endocannabinoid system using knockout ((-/-)) mice represent a powerful model but with some limitations due to compensation. To overcome such a limitation, we have generated an inducible and reversible tet-off dependent tissue-specific CB1(-/-) mice where the CB1R is re-expressed exclusively in the forebrain at a hypomorphic level due to a mutation (IRh-CB1(-/-)) only in absence of doxycycline (Dox). In such mice, under Dox(+) or vehicle, as well as in wild-type (WT) and CB1(-/-), two endophenotypes motor activity (increased in animal models of schizophrenia) and pre-pulse inhibition (PPI) of startle reflex (disrupted in schizophrenia) were analyzed. Both CB1(-/-) and IRh-CB1(-/-) showed increased motor activity when compared to WT animals. The PPI response, unaltered in WT and CB1(-/-) animals, was on the contrary highly and significantly disrupted only in Dox(+) IRh-CB1(-/-) mice. Such a response was easily reverted after either withdrawal from Dox or haloperidol treatment. This is the first Inducible and Reversible CB1(-/-) mice model to be described in the literature. It is noteworthy that the PPI disruption is not present either in classical full CB1(-/-) mice or following acute administration of rimonabant. Such a hypomorphic model may provide a new tool for additional in vivo and in vitro studies of the physiological and pathological roles of cannabinoid system in schizophrenia and in other psychiatric disorders.

A key regulatory gene in definitive erythropoiesis is the erythroid Kruppel-like factor (Eklf or Klf1). Klf1 knockout (KO) mice die in utero due to severe anemia, while residual circulating red blood cells retain their nuclei. Dnase2a is another critical gene in definitive erythropoiesis. Dnase2a KO mice are also affected by severe anemia and die in utero. DNase II-alpha is expressed in the central macrophage of erythroblastic islands (CMEIs) of murine fetal liver. Its main role is to digest the DNA of the extruded nuclei of red blood cells during maturation. Circulating erythrocytes retain their nuclei in Dnase2a KO mice. Here, we show that Klf1 is expressed in CMEIs and that it binds and activates the promoter of Dnase2a. We further show that Dnase2a is severely downregulated in the Klf1 KO fetal liver. We propose that this downregulation of Dnase2a in the CMEI contributes to the Klf1 KO phenotype by a non-cell-autonomous mechanism.

The CACCC box is duplicated in the beta-globin gene promoter of humans and other mammals. While the function of the proximal element as a binding site for EKLF has already been well established, the role of the distal element remains unclear. Mice present two adult beta-globin genes, beta-major and beta-minor, bearing a single CACCC box, the consensus sequence of which is identical to that of the proximal or distal human element, respectively. In the present study we analyzed the mRNA expression of beta-minor and beta-major in EKLF Knock-Out (KO) mice in comparison to wild-type (wt) littermates. The murine early fetal liver up to day 13/14 post coitum (pc) expresses mainly beta-minor globin chains. Nevertheless, expression of the beta-minor globin gene in EKLF KO mice has not been assessed to date. We provide evidence that expression of the beta-minor globin gene is dependent upon EKLF and is more affected by EKLF deprivation than the beta-major gene. The results obtained support a general role of EKLF in beta-globin gene activation and are in agreement with models involving an advantage of the LCR proximal respect to distal gene.

Genetic isolates with a history of a small founder population, long-lasting isolation and population bottlenecks represent exceptional resources in the identification of genes involved in the pathogenesis of multifactorial diseases. In these populations, the disease allele reveals linkage disequilibrium (LD) with markers over significant genetic intervals, therefore facilitating disease locus identification. This study has been designed to examine the background LD extension in some subpopulations of Corsica. Our interest in the island of Corsica is due to its geographical and genetic proximity to the other Mediterranean island of Sardinia. Sardinian isolates in which the extension of the background LD is particularly high have been recently identified and are now the object of studies aimed at the mapping of genes involved in complex diseases. Recent evidence has highlighted that the genetic proximity between the populations of Corsica and Sardinia is particularly true for the internal conservative populations. Given these considerations, Sardinia and Corsica may represent a unique system to carry out parallel association studies whose results could be validated by comparison. In the present study, we have analyzed the LD extension on the Xq13 genomic region in three subpopulations of Corsica: Corte, Niolo and Bozio, all located in the mountainous north-center of the island. Our results show a strong degree of LD over long distance for the population of Bozio and to a less extent for the population of Niolo. Their LD extent is comparable to or higher than that reported for other isolates.

The delta-globin gene produces the delta chain of Hb A2 which represents less than 3% of the hemoglobin (Hb) in normal individuals. The delta-globin gene is also expressed in the human erythroleukemia cell line K562. The expression of the delta-globin gene in this cell line is unexpected since K562 shows an embryonic-fetal globin gene expression pattern with no expression of the adult beta-globin gene. delta-Globin gene activation has been proposed as a potential therapeutic tool for the cure of delta-thalassemia (thal). In order to shed some light on the delta-globin gene activation in K562 the present study has: (1) determined the complete nucleotide sequence of the delta- and beta-globin genes; (2) assessed, by reverse transcription-polymerase chain reaction (RT-PCR), the relative delta- and beta-globin mRNA level; and (3) analyzed the exact level of the endogenous expression delta-globin gene by S1 mapping. No sequence variations were identified in the (delta- and beta-globin genes when compared to the normal sequences. delta-Globin mRNA represent more than 95% of the total delta + beta-mRNA content. The level of expression of the delta-globin gene is 12.3% (+/- 1.2) compared to the endogenous alpha-globin gene. These results indicate that the high expression of the delta-globin gene in K562 is most likely due to the transacting environment. Therefore, the presence and/or absence of specific transacting factors are able to specifically activate the human delta-globin gene. The level of expression of the delta-globin gene in this cell line suggests that it could be of relevance to identify the transacting factor(s) responsible for this selective activation in order to better understand the molecular mechanisms undergoing gene activation.

Objective: To determine if the extent of lymphadenectomy (number of recovered lymph nodes) was associated with long-term outcome in patients operated on for stage B and C colon cancer., Summary Background Data: Lymphatic spreading is the main prognostic indicator in colon cancer patients, although the optimal extent of lymphadenectomy and its prognostic impact are still unknown., Methods: In 3,648 patients (median follow-up 3.6 years) enrolled in two consecutive INTACC multicentric trials on adjuvant therapy for colon cancer, we studied the association of the number of recovered nodes with overall survival and relapse free survival by means of univariate and Cox regression analysis., Results: The worst overall survival was related to ages > 65 (risk ratio [RR] = 1.30), higher grading (RR = 1.96). Better overall survival was related to female gender (RR = 0.80) and to higher number of recovered nodes (8-12 nodes, RR = 0.46, 13-17 nodes, RR = 0.76, nodes > or = 18, RR = 0.79). The same pattern was observed for relapse free survival. Longer overall and relapse free survival were related to a higher number of recovered nodes with P =.034 and P =.003 respectively (stratified analysis for absence or presence of positive nodes). Stage B patients with fewer than 7 nodes in the specimen had both shorter overall survival (P =.0000) and relapse free survival (P =.0016) than the other B patients. Outcome of stage C patients was not related to the number of recovered nodes (P =.28 and 0.12 respectively). The interaction test between stage of disease and number of recovered nodes was statistically significant (P =.017)., Conclusions: Stage B patients with a small number of examined nodes may be understaged. Thus, these patients might be considered for adjuvant therapy because of their poorer life expectancy than other stage B patients. For stage C patients, the number of recovered nodes does not seem to affect long-term outcome.

During the switch from human gamma- (fetal) to beta- (adult) globin gene expression, the gamma and beta genes are expressed competitively by an alternating transcription mechanism. The -50 region of the gamma gene promoter has been proposed to be responsible for the early competitive advantage of the gamma genes and to act as a stage selector element (SSE) in hemoglobin switching. We analyzed the effect of mutating the -50 region of the gamma gene in the presence of a competing beta gene in transgenic mice. This shows that the -50 region does not affect silencing of the beta gene in early development and does not act as a stage selector. However, it affects the ratio of gamma versus beta gene expression in the early, but not later, stages of fetal development. Interestingly, both the wild-type and mutant minilocus constructs show a higher frequency of alternate transcription than observed in the complete locus, suggesting that sequences normally present between the gamma and beta genes facilitate the interaction of the locus control region (LCR) and beta-globin gene in the complete locus.

The rat tyrosine hydroxylase gene (TH) from a panel of outbred and inbred rat strains has been analysed by Southern blotting, restriction-endonuclease mapping and direct sequencing of PCR-amplified products for detecting DNA polymorphisms. Five polymorphic sites have been characterized. This information may be used in pharmacogenetic studies to determine the influence of the TH gene in animal models of affective disorders and addictive behaviours.

Aim of this study is to carry out a genetic analysis of polymorphisms of the renin-angiotensin system in a genetically homogeneous population, in patients with and without myocardial infarction (AMI) expansion and to evaluate the influence of non genetic, mechanical factors. The study was conducted on 299 patients with first AMI. Ecocardiography studies were performed on all patients on day 1 and 3 from the onset of AMI and before discharge. Eighty-four patients were excluded because of inadequate quality of echocardiograms and 215 (163 males, 52 females) were admitted. Of these, 157 had no evidence of AMI expansion (EXP-) while 58 had expansion (EXP+). DNA was extracted by standard methods from blood samples. Age and gender had no influence on AMI expansion. Anterior infarction (p < 0.000001) and Q-wave infarction (p < 0.00002) were found more frequently in EXP+. Peak of creatine phosphokinase was higher in EXP+ than in EXP- (p < 0.00001). The percent of patients treated with thrombolysis or with hypertension and/or left ventricular hypertrophy was not significantly different in the two groups. AGT MT235 polymorphism of angiotensinogen gene, I/D polymorphism of ACE gene and AT1 A1166C of AT1 receptor of angiotensin II were not significantly different in two groups. Stratified analysis showed that in patients with anterior AMI (n = 87), with a higher risk of AMI expansion, there is a significant difference (p < 0.02) in ACE genotype between EXP- and EXP+. Odds ratio assuming the dominant effect of I allele (II+ ID < DD) was 3.35 (confidence interval 1.41-7.56) with increased risk of expansion. More extension studies are need to verify if these results can contribute to early identification of patients at higher risk and to optimize therapeutic approach.

In this paper we report the molecular characterization of a large deletion that removes the entire Factor VIII gene in a severe hemophilia A patient. Accurate DNA analysis of the breakpoint region revealed that a large DNA fragment replaced the 300-kb one, which was removed by the deletion. Pulsed-field gel electrophoresis analysis revealed that the size of the inserted fragment is about 550 kb. In situ hybridization demonstrated that part of the inserted region normally maps to Xq21 and to the tip of the short arm of the Y chromosome (Yp). In our patient this locus is present both in Xq21 and in Xq28, in addition to the Yp, being thus duplicated in the X chromosome. Sequence analysis of the 3' breakpoint suggested that an illegitimate recombination is probably the cause of this complex rearrangement.

Aims: Twenty-three patients with liver metastases from colorectal cancer were entered into a prospective, phase II pilot study to evaluate the efficacy and feasibility of intra-arterial high-dose chemotherapy (IAHC) + intraperitoneal chemotherapy (IPC) combined with hemofiltration., Methods: All patients had abdominal laparotomy to position a hepatic artery infusion port and in 15 cases an implantable system for IPC. A double-lumen filtration catheter was placed in the vena cava via the saphenous or femoral vein and connected to a modified hemofiltration unit. The treatment schedule consisted of mitomycin (30-50 mg/m2) and epirubicin (60-90) mg/m2) as IAHC combined with cisplatin (60 mg/m2) given in a 2000 ml saline solution by IPC. The high-dose IAHC-IPC was followed by 4 cycles of intra-arterial standard dose chemotherapy through the arterial port-a-cath (6 mg/m2 mitomycin and 20 mg/m2 epirubicin) and if possible by another cycle of high dose IAHC-IPC., Results: We delivered a total of 31 cycles of IAHC, 21 of which were combined with IPC. Ten cycles of IAHC were administered without concurrent IPC because of painful adhesions, clinical contraindications or patient refusal. Seven of 23 patients (30%) were pretreated and with progressive disease after systemic chemotherapy. Among 22 evaluable patients, we obtained 2 complete remissions (9%) and 11 partial remissions (50%); moreover, 4 of 7 pretreated patients obtained a response to treatment. As a result, an objective tumor response was observed in 59% of patients (13/22). Therefore, a dose-response behavior was demonstrated also in tumors with a low chemosensitivity. The median duration of response and survival was 10 and 14 months, respectively. Toxicity was usually mild, but we reported one toxic death due to treatment complications., Conclusions: Further prospective randomized studies are needed to confirm the results of our study.

This study describes a patient with a thalassemia intermedia-like phenotype in whom beta-globin gene sequencing detected a novel abnormal hemoglobin (Hb) due to a T-C substitution at codon 114 of the beta-globin gene arising as a de novo mutation. The abnormal variant was designated Hb Brescia after the place of birth of the propositus. Normal sequences were detected at the in trans beta-globin locus. In addition, alpha-globin gene analysis detected a triple alpha-globin locus which was inherited from the father. The T-C change at position 114 of the beta-globin gene results in a leucine to proline substitution (Leu-Pro) in the G-helix. The resulting Hb tetramer is highly unstable and precipitates forming inclusion bodies in the peripheral red blood cells. Moreover, the Leu-Pro substitution interferes negatively with the four alpha 1 beta 1 contact points of the G-helix most likely adversely affecting the alpha beta dimer formation. The very severe phenotype presented by our patient is unusual in a heterozygote for an unstable Hb variant and may be explained by the coinheritance of the triple alpha-globin locus.

The authors report their experience concerning the cultural study of bile samples taken during elective biliary surgery. All the operations were carried out after a randomized preoperative antibiotic prophylaxis. Among the 138 cases examined the presence of surgical infections (wound infection) was taken into account. A lack of correlation between the germs present in the bile and those isolated from samples of complicated cases, was registered. However, the Authors emphasize that, although antibiotic prophylaxis gives a reduced percentage of postoperative infections, it is unable to sterilize the bile.

An assessment is made of the systematic application of intraoperative cholangiography in the light of a consecutive series gathered over a period of four years. The importance of employing this technique as a routine measure is clearly demonstrated by the results. If properly performed, it greatly reduces unnecessary choledochotomy, diminishes the incidence of overlooked calculi, and leads to the detection of papillo-oddian diseases.

The authors report their experience about splenic breakages in the blunt abdominal traumatology. Through the analysis of different parameters (type of accident, other associated lesions, heart frequency, hematocrit, paracentesis, direct radiological inspection of abdomen, gap of time between hospitalization and operation) they try to frame the blunt traumata of abdomen in four differential and orientative classes. As a conclusion; they assert the diagnosis of splenic breakage can be placed in any hospitals, with no need for sophisticated investigations, but following clinico-laboratorial criteria.