Prenatal Diagnosis of β-Thalassemias and Hemoglobinopathies.

Prenatal diagnosis of β-thalassemia was accomplished for the first time in the 1970s by globin chain synthesis analysis on fetal blood obtained by placental aspiration at 18–22 weeks gestation. Since then, the molecular definition of the β-globin gene pathology, the development of procedures of DNA analysis, and the introduction of chorionic villous sampling have dramatically improved prenatal diagnosis of this disease and of related disorders. Much information is now available about the molecular mechanisms of the diseases and the molecular testing is widespread. As prenatal diagnosis has to provide an accurate, safe and early result, an efficient screening of the population and a rapid molecular characterization of the couple at risk, are necessary prerequisites. In the last decades earlier and less invasive approaches for prenatal diagnosis were developed. A overview of the most promising procedure will be done. Moreover, in order to reduce the choice of interrupting the pregnancy in case of affected fetus, Preimplantation or Preconceptional Genetic Diagnosis (PGD) has been setting up for several diseases including thalassemia

Characterization of a disease-associated mutation affecting a putative splicing regulatory element in intron 6b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene

Cystic fibrosis (CF) is a common recessive disorder caused by >1600 mutations in the CF transmembrane conductance regulator (CFTR) gene. About 13% of CFTR mutations are classified as “splicing mutations,” but for almost 40% of these, their role in affecting the pre-mRNA splicing of the gene is not yet defined. In this work, we describe a new splicing mutation detected in three unrelated Italian CF patients. By DNA analyses and mRNA studies, we identified the c.1002–1110_1113delTAAG mutation localized in intron 6b of the CFTR gene. At the mRNA level, this mutation creates an aberrant inclusion of a sequence of 101 nucleotides between exons 6b and 7. This sequence corresponds to a portion of intron 6b and resembles a cryptic exon because it is characterized by an upstream ag and a downstream gt sequence, which are most probably recognized as 5′- and 3′-splice sites by the spliceosome. Through functional analysis of this splicing defect, we show that this mutation abolishes the interaction of the splicing regulatory protein heterogeneous nuclear ribonucleoprotein A2/B1 with an intronic splicing regulatory element and creates a new recognition motif for the SRp75 splicing factor, causing activation of the cryptic exon. Our results show that the c.1002–1110_1113delTAAG mutation creates a new intronic splicing regulatory element in intron 6b of the CFTR gene exclusively recognized by SRp75

AIRE acetylation and deacetylation: effect on protein stability and transactivation activity

The AIRE protein plays a remarkable role as a regulator of central tolerance by controlling the promiscuous expression of tissue-specific antigens in thymic medullary epithelial cells. Defects in AIRE gene cause the autoimmune polyendocrinopathy- candidiasis-ectodermal dystrophy, a rare disease frequent in Iranian Jews, Finns, and Sardinian population. AIRE protein is primarily known as a transcriptional regulator and is capable of interacting with numerous proteins. The first characterized partner of AIRE is the ubiquitous transcription factor CREB-binding protein (CBP), which regulates DNA transcription through the acetylation and deacetylation of histones. More recently, the role of p300 in AIRE acetylation, which could influence the selection of AIRE activated genes, has been described. Results: In this study, we have precisely mapped, by mass spectrometry experiments, the sites of protein acetylation and, by mutagenesis assays, we have described a set of acetylated lysines as being crucial in influencing the subcellular localization of AIRE. Furthermore, we have also determined that the de-acetyltransferase enzymes HDAC1-2 are involved in the lysine de-acetylation of AIRE. Conclusions: On the basis of our results and those reported in literature, we propose a model in which lysines acetylation increases the stability of AIRE in the nucleus. In addition, we observed that the interaction of AIRE with deacetylases complexes inhibits its transcriptional activity and is probably responsible for the instability of AIRE, which becomes more susceptible to degradation in the proteasome

Vitamin D responsive elements within the HLA-DRB1 promoter region in Sardinian multiple sclerosis associated alleles

Vitamin D response elements (VDREs) have been found in the promoter region of the MS-associated allele HLA-DRB1*15:01, suggesting that with low vitamin D availability VDREs are incapable of inducing *15:01 expression allowing in early life autoreactive T-cells to escape central thymic deletion. The Italian island of Sardinia exhibits a very high frequency of MS and high solar radiation exposure. We test the contribution of VDREs analysing the promoter region of the MS-associated DRB1 *04:05, *03:01, *13:01 and *15:01 and non-MS-associated *16:01, *01, *11, *07:01 alleles in a cohort of Sardinians (44 MS patients and 112 healthy subjects). Sequencing of the DRB1 promoter region revealed a homozygous canonical VDRE in all *15:01, *16:01, *11 and in 45/73 *03:01 and in heterozygous state in 28/73 *03:01 and all *01 alleles. A new mutated homozygous VDRE was found in all *13:03, *04:05 and *07:01 alleles. Functionality of mutated and canonical VDREs was assessed for its potential to modulate levels of DRB1 gene expression using an in vitro transactivation assay after stimulation with active vitamin D metabolite. Vitamin D failed to increase promoter activity of the *04:05 and *03:01 alleles carrying the new mutated VDRE, while the *16:01 and *03:01 alleles carrying the canonical VDRE sequence showed significantly increased transcriptional activity. The ability of VDR to bind the mutant VDRE in the DRB1 promoter was evaluated by EMSA. Efficient binding of VDR to the VDRE sequence found in the *16:01 and in the *15:01 allele reduced electrophoretic mobility when either an anti-VDR or an anti-RXR monoclonal antibody was added. Conversely, the Sardinian mutated VDRE sample showed very low affinity for the RXR/VDR heterodimer. These data seem to exclude a role of VDREs in the promoter region of the DRB1 gene in susceptibility to MS carried by DRB1* alleles in Sardinian patients

A role of BRCA1 and BRCA2 germline mutations in breast cancer susceptibility within Sardinian population

<p>Abstract</p> <p>Background</p> <p>In recent years, numerous studies have assessed the prevalence of germline mutations in <it>BRCA1 </it>and <it>BRCA2 </it>genes in various cohorts. We here extensively investigated the prevalence and geographical distribution of <it>BRCA1-2 </it>mutations in the entire genetically-homogeneous Sardinian population. The occurrence of phenotypic characteristics which may be predictive for the presence of <it>BRCA1-2 </it>germline mutations was also evaluated.</p> <p>Methods</p> <p>Three hundred and forty-eight breast cancer patients presenting a familial recurrence of invasive breast or ovarian carcinoma with at least two affected family members were screened for <it>BRCA1-2 </it>mutations by DHPLC analysis and DNA sequencing. Association of <it>BRCA1 </it>and <it>BRCA2 </it>mutational status with clinical and pathological parameters was evaluated by Pearson's Chi-Squared test.</p> <p>Results and Conclusion</p> <p>Overall, 8 <it>BRCA1 </it>and 5 <it>BRCA2 </it>deleterious mutations were detected in 35/348 (10%) families; majority (23/35;66%) of mutations was found in <it>BRCA2 </it>gene. The geographical distribution of <it>BRCA1-2 </it>mutations was related to three specific large areas of Sardinia, reflecting its ancient history: <it>a</it>) the Northern area, linguistically different from the rest of the island (where a <it>BRCA2 c.8764_8765delAG </it>mutation with founder effect was predominant); <it>b</it>) the Middle area, land of the ancient Sardinian population (where <it>BRCA2 </it>mutations are still more common than <it>BRCA1 </it>mutations); and <it>c</it>) the South-Western area, with many Phoenician and Carthaginian locations (where <it>BRCA1 </it>mutations are prevalent). We also found that phenotypic features such as high tumor grading and lack of expression of estrogen/progesterone receptors together with age at diagnosis and presence of ovarian cancer in the family may be predictive for the presence of <it>BRCA1-2 </it>germline mutations.</p

LIME -- a gas TPC prototype for directional Dark Matter search for the CYGNO experiment

The CYGNO experiment aims at the development of a large gaseous TPC with GEM-based amplification and an optical readout by means of PMTs and scientific CMOS cameras for 3D tracking down to O(keV) energies, for the directional detection of rare events such as low mass Dark Matter and solar neutrino interactions. The largest prototype built so far towards the realisation of the CYGNO experiment demonstrator is the 50 L active volume LIME, with 4 PMTs and a single sCMOS imaging a 33$\times$33 cm\textsuperscript{2} area for 50 cm drift, that has been installed in underground Laboratori Nazionali del Gran Sasso in February 2022. We will illustrate LIME performances as evaluated overground in Laboratori Nazionali di Frascati by means of radioactive X-ray sources, and in particular the detector stability, energy response and energy resolution. We will discuss the MC simulation developed to reproduce the detector response and show the comparison with actual data. We will furthermore examine the background simulation worked out for LIME underground data taking and illustrate the foreseen expected measurement and results in terms of natural and materials intrinsic radioactivity characterisation and measurement of the LNGS underground natural neutron flux. The results that will be obtained by underground LIME installation will be paramount in the optimisation of the CYGNO demonstrator, since this is foreseen to be composed by multiple modules with the same LIME dimensions and characteristics

Technical Design Report - TDR CYGNO-04/INITIUM

The aim of this Technical Design Report is to illustrate the technological choices foreseen to be implemented in the construction of the CYGNO-04 demonstrator, motivate them against the experiment physics goals of CYGNO-30 and demonstrate the financial sustainability of the project. CYGNO-04 represents PHASE 1 of the long term CYGNO roadmap, towards the development of large high precision tracking gaseous Time Projection Chamber (TPC) for directional Dark Matter searches and solar neutrino spectroscopy. The CYGNO project1 peculiarities reside in the optical readout of the light produced during the amplification of the primary ionization electrons in a stack of triple Gas Electron Multipliers (GEMs), thanks to the nice scintillation properties of the chosen He:CF4 gas mixture. To this aim, CYGNO is exploiting the fast progress in commercial scientific Active Pixel Sensors (APS) development for highly performing sCMOS cameras, whose high granularity and sensitivity allow to significantly boost tracking, improve particle identification and lower the energy threshold. The X-Y track project obtained from the reconstruction of the sCMOS images is combined with a PMT measurement to obtain a full 3D track reconstruction. In addition, several synergic R&Ds based on the CYGNO experimental approach are under development in the CYGNO collaboration (see Sec 2) to further enhance the light yield by means of electro luminescence after the amplification stage, to improve the tracking performances by exploiting negative ion drift operation within the INITIUM ERC Consolidator Grant, and to boost the sensitivity to O(GeV) Dark Matter masses by employing hydrogen rich target towards the development of PHASE 2 (see Sec. 1.2). While still under optimization and subject to possible significant improvements, the CYGNO experimental approach performances and capabilities demonstrated so far with prototypes allow to foresee the development of an O(30) m3 experiment by 2026 for a cost of O(10) MEUROs. A CYGNO-30 experiment would be able to give a significant contribution to the search and study of Dark Matter with masses below 10 GeV/c2 for both SI and SD coupling. In case of a Dark Matter observation claim by other experiments, the information provided by a directional detector such as CYGNO would be fundamental to positively confirm the galactic origin of the allegedly detected Dark Matter signal. CYGNO-30 could furthermore provide the first directional measurement of solar neutrinos from the pp chain, possibly extending to lower energies the Borexino measurement2. In order to reach this goal, the CYGNO project is proceeding through a staged approach. The PHASE 0 50 L detector (LIME, recently installed underground LNGS) will validate the full performances of the optical readout via APS commercial cameras and PMTs and the Montecarlo simulation of the expected backgrounds. The full CYGNO-04 demonstrator will be realized with all the technological and material choices foreseen for CYGNO-30, to demonstrate the scalability of the experimental approach and the potentialities of the large PHASE 2 detector to reach the expected physics goals. The first PHASE 1 design anticipated a 1 m3 active volume detector with two back-to-back TPCs with a central cathode and 500 mm drift length. Each 1 m2 readout area would have been composed by 9 + 9 readout modules having the LIME PHASE 0 dimensions and layout. Time (end of INITIUM project by March 2025) and current space availability at underground LNGS (only Hall F) forced the rescaling of the PHASE 1 active volume and design to a 0.4 m3, hence CYGNO-04. CYGNO-04 will keep the back-to-back double TPC layout with 500 mm drift length each, but with an 800 x 500 mm2 readout area covered by a 2 + 2 modules based on LIME design. The reduction of the detector volume has no impact on the technological objectives of PHASE 1, since the modular design with central cathode, detector materials and shieldings and auxiliary systems are independent of the total volume. The physics reach (which is a byproduct of PHASE 1 and NOT an explicit goal) will be only very partially reduced (less than a factor 2 overall) since a smaller detector volume implies also a reduced background from internal materials radioactivity. In addition, the cost reduction of CYGNO-04 of about 1⁄3 with respect to CYGNO-1 illustrated in the CDR effectively makes the overall project more financially sustainable (see CBS in the last section). In summary this document will explain: the physical motivation of the CYGNO project and the technical motivations of the downscale of the PHASE 1 to CYGNO-04, 400 liters of active volume, with respect to the demonstrator presented in the CDR; the results of R&D and the Montecarlo expectations for PHASE 0; the technical choices, procedures and the executive drawings of CYGNO-04 in the Hall F of the LNGS; safety evaluations and the interference/request to the LNGS services; Project management, WBS/WBC, WP, GANTT, ec

High Differentiation among Eight Villages in a Secluded Area of Sardinia Revealed by Genome-Wide High Density SNPs Analysis

To better design association studies for complex traits in isolated populations it's important to understand how history and isolation moulded the genetic features of different communities. Population isolates should not “a priori” be considered homogeneous, even if the communities are not distant and part of a small region. We studied a particular area of Sardinia called Ogliastra, characterized by the presence of several distinct villages that display different history, immigration events and population size. Cultural and geographic isolation characterized the history of these communities. We determined LD parameters in 8 villages and defined population structure through high density SNPs (about 360 K) on 360 unrelated people (45 selected samples from each village). These isolates showed differences in LD values and LD map length. Five of these villages show high LD values probably due to their reduced population size and extreme isolation. High genetic differentiation among villages was detected. Moreover population structure analysis revealed a high correlation between genetic and geographic distances. Our study indicates that history, geography and biodemography have influenced the genetic features of Ogliastra communities producing differences in LD and population structure. All these data demonstrate that we can consider each village an isolate with specific characteristics. We suggest that, in order to optimize the study design of complex traits, a thorough characterization of genetic features is useful to identify the presence of sub-populations and stratification within genetic isolates

The CYGNO Experiment

The search for a novel technology able to detect and reconstruct nuclear and electron recoil events with the energy of a few keV has become more and more important now that large regions of high-mass dark matter (DM) candidates have been excluded. Moreover, a detector sensitive to incoming particle direction will be crucial in the case of DM discovery to open the possibility of studying its properties. Gaseous time projection chambers (TPC) with optical readout are very promising detectors combining the detailed event information provided by the TPC technique with the high sensitivity and granularity of latest-generation scientific light sensors. The CYGNO experiment (a CYGNus module with Optical readout) aims to exploit the optical readout approach of multiple-GEM structures in large volume TPCs for the study of rare events as interactions of low-mass DM or solar neutrinos. The combined use of high-granularity sCMOS cameras and fast light sensors allows the reconstruction of the 3D direction of the tracks, offering good energy resolution and very high sensitivity in the few keV energy range, together with a very good particle identification useful for distinguishing nuclear recoils from electronic recoils. This experiment is part of the CYGNUS proto-collaboration, which aims at constructing a network of underground observatories for directional DM search. A one cubic meter demonstrator is expected to be built in 2022/23 aiming at a larger scale apparatus (30 m$^3$--100 m$^3$) at a later stage