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The triple A syndrome (AAAS) is an inherited condition associated with mutations in the AAAS gene, which encodes a protein of 546 amino acids known as ALADIN (alacrima achalasia adrenal insufficiency neurologic disorder) whose function is not well understood. This protein belongs to the WD-repeat family of regulatory proteins and is located in the nuclear pore complexes. Only a few cohorts of AAAS patients have been reported and fully characterized. Thus, the objective of the present study was to report on a mini cohort of Italian AAAS patients and to get insights on their predisposing genetic defects. Genetic analysis of AAAS gene in triple A syndrome patient and molecular and functional characterization of the novel identified allelic variants. Here we describe three newly diagnosed cases of AAAS, in whom genetic analysis allowed us to identify two novel allelic variants in the AAAS gene: the frameshift substitution c.765 dupT (p.Gly256Trp fsX67) in exon 8 and the splice site mutation in intron 11(c.997–2 A > G, IVS11-2A > G). Both variants result in a truncated non-functional protein, as we demonstrate by transcript analysis and expression studies. Our findings establish a pathogenic role for both new variants. Moreover, our data highlight the essential role of the C-terminal domain of the protein for its correct targeting and function and underline the importance of sequencing splice sites surrounding the intron–exon junctions to ensure accurate molecular diagnosis and correct genetic counseling in AAAS patients.

The main goal of this study was to identify Gram-negative bacteria resistant to antibiotics, in particular β-lactams, in stream waters and effluents from urban wastewater treatment plants draining into Fino, Tavo, and Saline rivers of the Abruzzo region, Italy. Eight sampling sites were selected because they were the most contaminated by coliforms during previous sampling campaign. One sample for each site was collected for the detection of total and fecal coliforms

SUMMARY The most important aspects of carrier detection procedures, genetic counselling, population screening and fetal diagnosis of the thalassaemias and sickle cell anaemia are reviewed. Carrier detection can be made retrospectively, i.e. following the birth of an affected child, or prospectively. Most carrier detection and genetic counselling in population at risk for alpha-thalassaemia an sickle cell anaemia is retrospective. However, some prospective carrier screening programmes for sickle cel anaemia are ongoing in Cuba and Guadeloupe and very limited screening for alpha-thalassaemia is in progress in some South East Asian populations. As regards beta-thalassaemia, several programmes, based on carrier screening and counselling of couples at marriage, preconception, or early pregnancy, have been operating with several populations at risk in the Mediterranean. These programmes have been very effective, as is proved by the fact that the target population has improved its knowledge of thalassaemia and its prevention, and by the marked decline that has been observed in the incidence of thalassaemia major. Carrier detection is carried out by haematological methods, followed by mutation detection by DNA analysis. Prenatal diagnosis is accomplished by mutation analysis on PCR-amplified DNA from chorionic villi. Future prospects include automation of the process of mutation detection, simplification of preconception and preimplantation diagnosis, and fetal diagnosis by analysis of fetal cells in the maternal circulation. In this article The most important aspect of carrier detection procedures, genetic counselling, population screening and fetal diagnosis of the thalassaemias and sickle cell anaemia are reviewed

A series of secondary and tertiary aryl sulfonamides were synthesized under flow conditions and evaluated for their ability to selectively inhibit tumor-associated carbonic anhydrase isoforms IX and XII. The tested compounds revealed to be highly potent CA IX inhibitors in nanomolar range, and to inhibit CA XII activity with different ranks of potencies. Remarkably, 4-methyl-N-phenyl-benzenesulfonamide was a selective nanomolar CA IX inhibitor with an IC50 of 90 nM.

Background: β-Thalassemia is one of the most common genetic diseases in humans. We developed an automated electronic microchip for fast and reliable detection of the nine most frequent mutations accounting for >95% of the β-thalassemia alleles in the Mediterranean area. Methods: We developed a microchip-based assay to identify the nine most frequent mutations (cd39C>T, IVS1-110G>A, IVS1-1G>A, IVS1-6T>C, IVS2-745C>G, cd6delA, −87C>G, IVS2-1G>A, and cd8delAA) by use of the Nanogen Workstation. The biotinylated amplicon was electronically addressed on the chip to selected pads, where it remained embedded through interaction with streptavidin in the permeation layer. The DNA at each test site was then hybridized to a mixture of fluorescently labeled wild-type or mutant probes. Results: Assays conditions were established based on the analysis of 700 DNA samples from compound heterozygotes or homozygotes for the nine mutations. The assays were blindly validated on 250 DNA samples previously genotyped by other methods, with complete concordance of results. Alternative multiplexed formats were explored: the combination of multiplex PCR with multiple addressing and/or hybridization allowed analysis of all nine mutations in the same sample on one test site of the chip. Conclusions: The open flexible platform can be designed by the user according to the local prevalence of mutations in each geographic area and can be rapidly extended to include the remaining mutations causing β-thalassemia in other regions of the world.

Since 2001 the Istituto Superiore di Sanità established a quality assurance programme for molecular genetic testing that covers four pathologies: Cystic Fibrosis (CF), Beta Thalassemia (BT), Fragile X Syndrome (FX), and Familial Adenomatous Polyposis Coli (APC). Since 2009 this activity is an institutional activity and participation is open to both public and private laboratories. Seven rounds have been performed until now and the eighth is in progress. Laboratories receive 4 DNA samples with mock clinical indications. They analyze the samples using their routine procedures. A panel of assessors review the raw data and the reports; all data are managed through a web utility. In 2010 the number of participants was 43, 17, 15, 5 for CF, BT, FX, APC schemes respectively. Genotyping results were correct in 96%, 98.5%, 100%, and 100% of CF, BT, FX, and APC samples, respectively. Interpretation was correct in 74%, 91%, 88%, and 60% of CF, BT, FX, and APC reports, respectively; however in most of them it was not complete but a referral to genetic counseling was given. Reports were satisfactory in more than 60% of samples in all schemes. This work presents the 2010 results in detail comparing our data with those from other European schemes.

Cystic fibrosis (CF) is a common recessive genetic disease caused by mutations in the gene encoding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. More than 1800 different mutations have been described to date. Here, we report 3 novel mutations in CFTR in 3 Italian CF patients. To detect and identify 36 frequent mutations in Caucasians, we used the INNO-LiPA CFTR19 and INNO-LiPA CFTR17+Tn Update kits (Innogenetics; Ghent, Belgium). Our first analysis did not reveal both of the responsible mutations; thus, direct sequencing of the CFTR gene coding region was performed. The 3 patients were compound heterozygous. In one allele, the F508del (c.1521_1523delCTT, p.PHE508del) mutation in exon 11 was observed in each case. For the second allele, in patient No.1, direct sequencing revealed an 11-base pair deletion (GAGGCGATACT) in exon 14 (c.2236_2246del; pGlu746Alafs*29). In patient No. 2, direct sequencing revealed a nonsense mutation at nucleotide 3892 (c.3892G>T) in exon 24. In patient No. 3, direct sequencing revealed a deletion of cytosine in exon 27 (c.4296delC; p.Asn1432Lysfs*16). These 3 novel mutations indicate the production of a truncated protein, which consequently results in a non-functional polypeptide.

β-Thalassemia is the most common autosomal recessive single-gene disorder in Sardinia, where approximately 10.3% of the population is a carrier. Prenatal diagnosis is carried out at 12 weeks of gestation via villocentesis and is commonly aimed at ascertaining the presence or absence of the HBB variant c.118C>T, which is the most common in Sardinia. In this study, we describe for the first time the application of semiconductor sequencing to the non-invasive prenatal diagnosis of β-thalassemia in 37 couples at risk for this variant. In particular, by using a haplotyping-based approach with a hidden Markov model (HMM) and a dedicated pipeline, the two parental haplotypes most likely inherited by the foetus could be established in 30 out of 37 cffDNA samples. Specifically, the paternally inherited haplotype was correctly determined in all 30 of the samples, while the maternal haplotype was incorrectly predicted in six of the 30 genotyped samples. The lack of informative SNPs hampered the inference of both parental haplotypes in the remaining seven samples. As shown in previous studies, we have confirmed that the haplotyping-based approach represents a valuable resource, as it improves the detection of both parental haplotypes inherited by the foetus. In general, our results are encouraging, as we have proven that NIPD is also feasible in couples who are at risk for a monogenic disorder and share the same variant.

Objective he aim of this study was to investigate whether a variation in the genomic copy number (CNV) of the β-defensin cluster could be associated with the predisposition to Chronic Mucocutaneous Candidiasis (CMC) in Sardinian APECED patients. Subjects and methods The β-defensin copy number variation was determined by MLPA analysis in 18 Sardinian APECED patients with CMC and in 21 Sardinian controls. Statistical analyses were performed with ANOVA-one way test. Results No statistically significant results were observed between the patients and controls groups. Conclusions According to the results we have obtained, it appears that either β-defensin genomic CNV is not a modifier locus for CMC susceptibility in APECED patients, or that any effect is too small for it to be detected using such sample size. An extensive study on APECED patients from different geographical areas might reveal the real implication of the β-defensin CNV in the susceptibility to Candida albicans infections. This article is protected by copyright. All rights reserved.

Autoimmune polyendocrine syndrome type 1 (APS1) is a childhood-onset monogenic disorder caused by mutations in the autoimmune regulator (AIRE) gene, including the distinctive R139X in Sardinia. Its rarity and great variability in manifestations/onset ages make early diagnosis difficult. To date, very few longitudinal studies of APS1 patients have been reported.The aim of this study was to describe the features and clinical course of APS1 and correlate them with AIRE and HLA class II genotypes in a large homogeneous cohort of Sardinian patients followed for up to 25 yr.Twenty-two pediatric APS1 patients were studied prospectively.This Sardinian series (female/male ratio, 1.44; median current age, 30.7 yr; range, 1.8-46 yr) showed early disease onset (age range, 0.3-10 yr; median, 3.5 yr) and severe phenotype (on average, seven manifestations per patient). Besides the classic triad of chronic mucocutaneous candidiasis, hypoparathyroidism, and Addison's disease, autoimmune hepatitis was a serious and surprisingly common/early/presenting feature (27%; two deaths), with a 5:1 female bias (median age, 6 yr; range, 2.5-11 yr). By contrast, type 1 diabetes was rare (one patient), and hypothyroidism was not seen. Additional disease components (several of them potentially life-threatening) appeared in adulthood. The major nonsense mutation, R139X, was found in 93% of the mutant AIRE alleles. High-titer interferon (IFN)-ω and IFN-α autoantibodies were detected in all patients tested, even preclinically at 4 months of age in one sibling. HLA alleles appear to influence the exact phenotype-the most interesting apparent association being between HLA-DRB1*0301-DQB1*0201, liver-kidney microsome autoantibodies (anti-CYP1A2), and autoimmune hepatitis.APS1 in Sardinia is characterized by severe phenotype, marked clinical heterogeneity, and relative genetic homogeneity. The single AIRE mutation, R139X, and the anti-IFN-ω and IFN-α autoantibodies are helpful for earlier diagnosis, especially when APS1 presents unusually. HLA genotypes can modify the phenotype.

Background: In Sardinia the mutational spectrum of CFTR gene is well defined. A mutation detection rate of 94% can be achieved by screening for 15 CFTR mutations with a frequency higher than 0.5%. The efficiency of this molecular test suggests that Sardinians may represent a suitable population for a preconceptional screening. Methods: Five hundred couples of Sardinia descent were screened for 38 mutations using a semi-automated reverse-dot blot and PCR-gel electrophoresis assays. This mutation panel included the 15 most frequent CF alleles in Sardinia. Results: We identified 38 CF carriers, revealing an overall frequency of 1/25 (4%). The most common CF allele was the p.Thr338Ile (T338I) (65%), followed by the p.Phe508del (F508del) (22.5%). We also identified one couple at risk and an asymptomatic female homozygote for the p.Thr338Ile allele. Conclusions: In spite of the low number of the couples tested, the results herein reported demonstrate the efficacy and efficiency of the preconceptional screening program and the high participation rate of the Sardinian population (99%).

In the framework of the design and development of TGR5 agonists, we reported that the introduction of a C(23)(S)-methyl group in the side chain of bile acids such as chenodeoxycholic acid (CDCA) and 6-ethylchenodeoxycholic acid (6-ECDCA, INT-747) affords selectivity for TGR5. Herein we report further lead optimization efforts that have led to the discovery of 6alpha-ethyl-23(S)-methylcholic acid (S-EMCA, INT-777) as a novel potent and selective TGR5 agonist with remarkable in vivo activity.

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.

The Italian scheme of External Quality Assessment for beta-thalassemia started in 2001 as part of a project twice funded by the Italian Ministry of Health and coordinated by the Istituto Superiore di Sanità. To date, five trials have been performed (2001-2004 and 2006). The aim of the Italian scheme is to help public laboratories in improving and reaching a high standard of quality when performing a molecular test. Many laboratories took part in the 5-year project, and their participation was constant during the whole period. The aims of this paper are to describe the genotyping and reporting results as well as focusing on the techniques and the testing strategies adopted to detect mutations. Almost 99% of the alleles analyzed were correctly detected by laboratories, while 0.33% of the analyses gave a wrong result. Reverse dot blot was the most used technique, and it was always used in the strategies adopted by laboratories to detect mutations. The reports sent by laboratories showed incompleteness and heterogeneity; thus, a new model for written reports has been introduced since 2004. It will be interesting to monitor the effects of the reporting model and the output of this educational action in the future.

To investigate the bronchiolitis-associated encephalopathy in critically ill infants.The records of infants with severe bronchiolitis admitted to our intensive care unit between 1991 and 2003 were reviewed. Subjects with underlying neurological disorders were excluded. Encephalopathy was defined as occurrence of seizures or at least two nonconvulsive neurologic manifestations. A semistructured telephone interview investigated long-term neurodevelopmental outcome.Twenty-one infants (11 newborns) were enrolled. All patients required oxygen supplementation and 14 required mechanical ventilation. Encephalopathy occurred in 10 infants, six of whom developed seizures. Encephalopathic infants frequently (six of nine) showed transient EEG abnormalities, and occasionally (one of nine) cranial ultrasound abnormalities. A positive respiratory syncytial virus test was found in five of nine encephalopathic infants. One encephalopathic patient died, while 20 infants clinically normalised before discharge and showed a good neurodevelopmental outcome.Acute encephalopathy was frequently observed in our patients with severe bronchiolitis. Long-term prognosis of encephalopathic infants was good.

This paper describes the first case of Hb Bart's hydrops fetalis syndrome in the Sardinian population. Despite the high frequency of a-thalassemia, fetal hydrops is extraordinarily rare in the Sardinian population because a-thalassemia is more usually the result of the single a-thalassemia globin gene deletion and is very rarely produced by the deletion of two a-globin genes. The fetus, the product of a consanguineous marriage at risk for beta-thalassemia, was monitored by chorionic villi DNA analysis which detected the heterozygous state for the codon 39 nonsense mutation. Follow-up ultrasound examination showed fetal hydrops, which led us to carry out further investigation. Hemoglobin and a-globin gene analysis on cord blood obtained by cordocentesis revealed the homozygous state for the most common deletion ao-thalassemia in Mediterranean populations. Retrospective evaluation of the father's hematological features showed very low MCH-MCV for a beta-thalassemia carrier which may indicate co-inherited a-thalassemia. These findings indicate that careful evaluation of red cell indices of parents at risk for beta-thalassemia and adequate consideration of the consanguinity may point to co-inherited a-thalassemia and lead to the appropriate analysis.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare autosomic autoimmune disease resulting from the defective function of a gene codifying for a transcription factor named autoimmune regulation (AIRE). The AIRE protein contains several domains among which two PHD fingers involved in the transcriptional activation. We investigated the function of the two PHD finger domains and the COOH terminal portion of AIRE by using several mutated constructs transfected in mammalian cells and a luciferase reporter assay. The results predict that the second PHD as well as the COOH terminal regions have marked transactivational properties. The COOH terminal region contains the fourth LXXLL and the PXXPXP motifs which play a critical role in mediating the transactivation capacity of the AIRE protein. Our study provides a definition of the role of the PHD fingers in transactivation and identifies a new transactivation domain of the AIRE protein localized in the COOH terminal region.

Jeune asphyxiating thoracic dystrophy (JATD; Jeune syndrome, MIM 208500) is a rare autosomal recessive chondrodysplasia, phenotypically overlapping with short-rib polydactyly syndromes (SRPS). JATD typical hallmarks include skeletal abnormalities such as narrow chest, shortened ribs, limbs shortened bones, extra fingers and toes (polydactyly), as well as extraskeletal manifestations (renal, liver and retinal disease). To date, disease-causing mutations have been found in several genes, highlighting a marked genetic heterogeneity that prevents a molecular diagnosis of the disease in most families. Here, we report the results of whole-exome sequencing (WES) carried out in four JATD cases, belonging to three unrelated families of Sardinian origin. The exome analysis allowed to identify mutations not previously reported in the DYNC2H1 (MIM 603297) and WDR60 (MIM 615462) genes, both codifying for ciliary intraflagellar transport components whose mutations are known to cause Jeune syndrome.

A series of β-amino acids with lipophilic diaromatic side chain was synthesized and characterized pharmacologically on mouse γ-amino butyric acid (GABA) transporter subtypes mGAT1-4 in order to investigate structure activity relationships (SAR) for mGAT2 (corresponding to hBGT-1). Variation in the lipophilic diaromatic side chain was probed to understand the role of the side chain for activity. This yielded several selective compounds of which the best (1R,2S)-5a was more than 10 fold selective towards other subtypes, although potency was moderate. A docking study was performed to investigate possible binding modes of the compounds in mGAT2 suggesting a binding mode similar to that proposed for Tiagabine in hGAT1. Specific interactions between the transporter and the amino acid part of the ligands may account for a reverted preference towards mGAT2 over mGAT1.

Previous studies performed on Sardinian patients affected by cystic fibrosis (CF) have led to the identification of molecular defects in 87 of 88 patients. Two mutations, the F508del and T338I, were quite prevalent and accounted for 50% and 20% of the molecular defects, respectively. T338I has been detected rarely in other populations, most likely because of the genetic isolation of Sardinians. In the present study, we have performed a molecular analysis of the CF gene in eight Sardinian patients in whom only a single mutation has been defined. Using DNA analyses (Southern blot, single nucleotide polymorphisms, microsatellite analyses, and Extra-Long polymerase chain reaction) selected to detect gross gene rearrangement and by using mRNA studies, we detected a novel mutation c.54-5811_164 + 2186del8108ins182 in six of the eight patients investigated. This mutation consists of a gross deletion of 8108 bp spanning exon 2 with an insertion of 182 bp at the deletion junction, between nucleotide 54-5811 of intron 1 (IVS1 nt16864) and nucleotide 164 + 2186 of intron 2 (IVS2 nt 2186). By including the novel mutation in our mutation panel we are now able to reach a 95% detection rate, thereby improving the process of carrier detection and genetic counseling in Sardinia.

Anatomical study of the interosseous region of the sacroiliac joint (SIJ) complex.To document and quantify the surface topography of the interosseous region of the SIJ.A review of the literature reveals that little consideration has been given to the interosseous region of the SIJ anatomically, biomechanically, and clinically.The interosseous region of 11 cadaveric specimens (9 formalin embalmed and 2 fresh frozen) were studied. Ten specimens were 55 years of age or older and 1 was 20 years old. To view the interosseous surfaces of the sacrum and ilium the specimens were either axially sectioned (1-cm slices) or disarticulated. One fresh-frozen and 6 embalmed specimens were disarticulated and the remainder axially sectioned. The topography (surface ridging and areas of ossification) of the interosseous region was documented in all specimens and in 2 specimens the surfaces were 3-dimensionally reconstructed using modeling and animation software (MAYA; Autodesk, Inc, San Rafael, CA).Surface characteristics of the SIJ complex observed in specimens 55 years of age or older included moderate to extensive ridging of the interosseous region of the sacrum and ilium in 100% of specimens and ossification of the central interosseous region of the sacroiliac (SI) ligament in 60% of specimens.Central region ossification of the interosseous SI ligament and the presence of ridges and depressions over the opposing interosseous surfaces of the sacrum and ilium are features common to specimens that are in or beyond their sixth decade. These findings further support the contention that there is little to no movement available at this joint in older individuals.

Background 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.

Pruritic papular eruption (PPE) is a common inflammatory cutaneous lesion observed only in HIV/AIDS patients. Since kinin is an important mediator in inflammation, we evaluated the levels of total kininogen (TKg), low and high molecular weight kininogen (LKg and HKg, respectively) and the activity of kallikrein in plasma of 11 patients (median age = 31.4) with AIDS and PPE (PPE+), eight patients (median age = 31.5) with AIDS without PPE (PPE-) and in 12 control individuals (median age = 32.9) with anti-HIV negative serum. Kininogens were measured by ELISA and expressed in median (m) of BK Equivalent/ml plasma and the kallikrein by its activity upon selective chromogenic substrate, and expressed as U kallikrein/ml of plasma. TKg or LKg concentrations in PPE+ patients (m = 4.11 and 4.5) and in PPE- patients (m = 6.23 and 4.54) were significantly higher when compared to control (m = 2.10 and 1.17). Compared to controls PPE- patients presented similar values of HKg (m = 0.78 and 0.61), whereas PPE+ patients presented undetectable values. Plasma kallikrein activity was significantly decreased in PPE+ and PPE- (m = 0.6 and 0.89, respectively) when compared with control individuals (m = 2.23).

The chemokine receptor CCR5 is encoded by the CMKBR5 gene located on the p21.3 region of human chromosome 3, and constitutes the major co-receptor for the macrophage-tropic strains of HIV-1. A mutant allele of the CCR5 gene, Delta ccr5 , was shown to provide to homozygotes with a strong resistance against infection by HIV. The frequency of the Delta ccr5 allele was investigated in 18 European populations. A North to South gradient was found, with the highest allele frequencies in Finnish and Mordvinian populations (16%), and the lowest in Sardinia (4%). Highly polymorphic microsatellites (IRI3.1, D3S4579 and IRI3.2, D3S4580 ) located respectively 11 kb upstream and 68 kb downstream of the CCR5 gene deletion were used to determine the haplotype of the chromosomes carrying the Delta ccr5 variant. A strong linkage disequilibrium was found between Delta ccr5 and specific alleles of the IRI3.1 and IRI3.2 microsatellites: >95% of the Delta ccr5 chromosomes carried the IRI3.1-0 allele, while 88% carried the IRI3.2-0 allele. These alleles were found respectively in only 2 or 1.5% of the chromosomes carrying a wild-type CCR5 gene. From these data, it was inferred that most, if not all Delta ccr5 alleles originate from a single mutation event, and that this mutation event probably took place a few thousand years ago in Northeastern Europe. The high frequency of the Delta ccr5 allele in Caucasian populations cannot be explained easily by random genetic drift, suggesting that a selection advantage is or has been associated with homo- or heterozygous carriers of the Delta ccr5 allele.

This study reports the molecular characterisation of the bilirubin UDP-glucuronosyl-transferase gene (UGT1) in a group of patients of Sardinian descent with Crigler-Najjar syndrome type I and their relatives. Sequence analysis of both UGT1A exon 1 and common exons 2-5 was performed in all patients, leading to the detection of AF170 and a novel mutation (470insT), both residing in UGT1A exon 1. All but two heterozygotes for the AF170 mutation showed normal serum bilirubin levels. These two subjects were also heterozygous for the sequence variation A(TA)7TAA in the promoter region of the UGT1A gene.

This paper reports a new hemoglobin variant which was identified while investigating the cause of a mild erythrocytosis. The abnormal beta-globin chain was detected by reversed phase chromatography. Mutation mapping of the beta-globin gene by polymerase chain reaction and denaturing gradient gel electrophoresis followed by sequence analysis revealed a C--A transversion at codon 38, predicting a Thr--Asn substitution. Tryptic peptide mapping by liquid chromatography electrospray mass spectrometry, followed by conventional Edman peptide sequence analysis, confirmed the predicted amino acid substitution. In contrast to the only other known mutation at codon 38, Hb Hazebrouck (Thr--Pro), this hemoglobin is stable and shows elevated oxygen affinity.

The G protein-coupled receptor TGR5 is a key player of the bile acid signaling network, and its activation has been proved to increase the glycemic control, to enhance energy expenditure and to exert anti-inflammatory actions. Accordingly, TGR5 ligands have emerged in drug discovery and preclinical appraisals as promising agents for the treatment of liver diseases, metabolic syndrome and related disorders.Recent advances in the field of TGR5 modulators are reviewed, with a particular attention on patent applications and peer-reviewed publications in the past 6 years.Activation of TGR5 showed to protect mice from diabesity and insulin resistance, to improve liver functions, as well as to attenuate the development of atherosclerosis. However, although the efficacy of TGR5 agonists in mice is encouraging, further studies are needed to determine their potential in humans and to evaluate carefully the balance between the therapeutic benefits and adverse effects associated with the target. The development of new TGR5 selective ligands to support studies in animal models will surely facilitate the understanding of the complexity of TGR5 signaling network.

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.

This paper describes the phenotypic manifestations of a very mild beta-thalassaemia mutation detected in several members of two families of Italian descent. The molecular defect, defined by denaturing gradient gel electrophoresis analysis and direct sequencing, consists of a C-->G substitution at position 844 of IVSII of the beta-globin gene within the consensus sequence of the IVSII acceptor splice site. Heterozygotes for this mutation show a haematological phenotype ranging in severity from silent beta-thalassaemia to that of a mild beta-thalassaemia carrier state, whereas homozygotes have the typical manifestations commonly resulting from heterozygosity for a beta-thalassaemia mutation. Compound heterozygotes for the IVSII nt844 (C-->G) mutation and a severe beta-thalassaemia mutation have the phenotype of thalassaemia intermedia. This paper indicates that the presence of borderline red blood cell indices or HbA2 values should make one suspect the presence of a very mild or silent beta-thalassaemia.

In this paper we review the molecular basis of the marked heterogeneity of the thalassemia syndromes as well as the relative implications for carrier screening and prenatal diagnosis. The classical phenotype of heterozygous beta-thalassemia may be modified by a number of environmental and genetic interacting factors--among which the most relevant are: (1) coinheritance of alpha-thalassemia, which may normalize the red blood cell indices; (2) the presence of a mild beta-thalassemia mutation; (3) cotransmission of delta-thalassemia which may reduce the increase of HbA2 typical of heterozygous beta-thalassemia to normal values and (4) the presence of a silent mutation which can be defined only by imbalanced beta-globin chain synthesis. A number of molecular mechanisms are able to produce the non transfusion dependent attenuated forms of thalassemia syndromes referred to as thalassemia intermedia. The most common are homozygosity for mild beta-thalassemia mutations, coinheritance with homozygous beta-thalassemia of alpha-thalassemia or genetic determinants able to sustain a continuous production of HbF in adult life or the presence of heterozygosity for hyperunstable globin variants.

Mutations within exons are responsible for aberrant splicing of pre-mRNA in several human disease genes and in some viral systems. Nonsense, missense, and even synonymous mutations can induce aberrant skipping of the mutant exon, producing nonfunctional proteins. In this paper, we describe the effect on the splicing efficiency of the synonymous variant 2811 G>T [Gly893Gly] detected in a patient of Italian descent affected by a mild form of cystic fibrosis, until now mentioned as sequence variation with unknown functional consequences. The study, performed through DNA as well as RNA analyses, shows that this mutation creates a new 5′ splice site within exon 15, resulting in a transcript lacking 76 amino acid residues. Although this aberrant splicing causes a shorter exon 15, the downstream exonic sequence from exon 16 to the end of the open reading frame is in frame. This study indicates that apparently neutral polymorphism, which may be erroneously classified as nonpathogenic, may indeed led to aberrant splicing thereby resulting in defective protein.

Background. Alport syndrome is a clinically and genetically heterogeneous nephropathy characterized by glomerular basement membrane lesions often associated with hearing loss and ocular anomalies. While the X-linked and the autosomal recessive forms are well known, the autosomal dominant form is not well acknowledged. Methods. We have clinically investigated 38 patients with a diagnosis of autosomal dominant Alport syndrome belonging to eight different families. The analysis of the COL4A4 gene was performed by denaturing high performance liquid chromatography and automated DNA sequencing. Results. In our cohort of patients, only 24.3% (9/37) reached end-stage renal disease, at the mean age of 51.2 Correspondence and offprint requests to: Alessandra Renieri, Medical Genetics, Department of Molecular Biology, University of Siena, VLe Bracci, 53100 Siena, Italy. Tel: +39-0577-233303; Fax: +39-0577- 233325; E-mail: renieri@unisi.it years. Four patients had hearing loss (13.3%) and none ocular changes. Molecular analysis revealed eight novel private COL4A4 gene mutations: three frameshift, three missense and two splice-site mutations. Conclusions. These data indicate autosomal dominant Alport syndrome as a disease with a low risk of ocular and hearing anomalies but with a significant risk to develop renal failure although at an older age than the X-linked form. We were unable to demonstrate a genotype-phenotype correlation. Altogether, these data make difficult the differential diagnosis with the benign familial haematuria due to heterozygous mutations of COL4A4 and COL4A3, especially in young patients, and with the X-linked form of Alport syndrome in families where only females are affected. A correct diagnosis and prognosis is based on a comprehensive clinical investigation in as many family members as possible associated with a broadly formal genetic analysis of the pedigree.

A preventive genetic programme aimed to control beta-thalassemia in the Sardinian population is based on a combination of increased awareness of the population, carrier screening, genetic counselling and prenatal diagnosis. As a result, the registry of thalassemia major demonstrated a profound decline in the incidence of this disease from 1 per 250 to 1 per 1200 live births, with 90% of cases effectively prevented.

This paper describes the manifestation in a child of a new syndrome characterized by unusual, severe, persistent hyponatremia associated with hyposmolarity, euvolemia, inappropriately concentrated urine and elevated natriuresis. This is the fourth case of this syndrome reported to date, and the first to be reported in a neonate. The clinical features resemble those typically observed in patients with inappropriate antidiuretic hormone secretion, although high arginine vasopressin (AVP) levels are lacking. The findings led the authors to hypothesise a nephrogenic syndrome of inappropriate antidiuresis (NSIAD). The previously described R137C gain-of-function mutation was detected by means of mutation analysis of the V2R gene. Our results indicate that NSIAD is already present during the neonatal period and that molecular analysis of the V2R receptor should therefore be carried out, in all newborns with prolonged euvolemic hyponatremia with hypo-osmolarity, high urinary sodium and normal/low or undetectable AVP levels.

This paper reviews the characteristics and the results of 15 years of experience with a preventive program, based on carrier screening and prenatal diagnosis, designed to control thalassemia major in the Sardinian population. The education of the population about thalassemia and the modalities for its prevention was accomplished via the mass media. Carrier screening was carried out voluntarily on couples of child-bearing age. Prenatal diagnosis was initially carried out by fetal blood analysis; since 1983, it has been done by DNA analysis on non-amplified or amplified DNA. Different chorionic villous sampling procedures have been used. Nowadays, we have adopted the transabdominal approach because, in our experience, it seems to be associated with a low risk (2%) of fetal mortality. At the present time, the beta-thalassemia mutations are detected directly by dot-blot analysis of amplified DNA with 32P- or horseradish peroxidase-labeled allele-specific oligonucleotide probes. Two oligonucleotide probes, one complementary to the codon-39 nonsense mutation, which accounts for 95.7% of the beta-thalassemia chromosomes in the Sardinian population, and the other complementary to the frameshift at codon 6, which is the second most common mutation in our population (2.1%), allow us to make prenatal diagnosis in the large majority of cases. Notwithstanding a careful dissection of maternal decidua from chorionic villi, co-amplification of maternal sequence was detected in 4 out of 425 cases tested by this procedure. In order to avoid this pitfall, the simultaneous amplification of highly polymorphic VNTR (variable number of tandem repeats) segments could be used. On the whole we have so far carried out 2711 prenatal tests: 1130 by fetal blood analysis, 1156 by oligonucleotide hybridization on electrophoretically separated DNA fragments, and 425 by dot-blot analysis on amplified DNA with allele-specific oligonucleotide probes. Two errors occurred by fetal blood analysis and none by DNA analysis. The incidence of thalassemia major declined from 1:250 live births in the absence of prevention to 1:1000 after the establishment of this program, indicating that carrier screening and prenatal diagnosis are effective means for preventing thalassemia major at the population level.

The Italian External Quality Control Programme for cystic fibrosis molecular diagnosis started in 2001; public laboratories distributed throughout Italy participated on a voluntary basis.: The Italian Public Health Institute (Istituto Superiore di Sanità) sent six validated DNA samples to participating laboratories: technical and clinical information was provided for each sample. Laboratories were required to analyse all six samples. For each sample the laboratories had to provide the results (including raw data) and a report of molecular analysis within 2 months using current methods and nomenclature. Raw data and reports were evaluated by a Steering Committee and their comments were sent to each laboratory.: Genotyping results indicated a general good level of quality for all laboratories, i.e., ∼1% of alleles were incorrectly assigned each year due to analytical (45%) and misinterpretation (45%) errors. During the first 2 years, more than 70% of laboratories did not test for some regional Italian mutations. Commercial kits for reverse dot-blot and oligonucleotide ligation assay PCR were used to detect mutations by 52.8% and 29.5%, respectively, of the participating laboratories. Reporting of results was still inadequate; in 2004 a model for the written report was introduced, but not all laboratories used it.: Our data show that few genotyping errors were made by laboratories and were principally due to misinterpretation and analytical reasons. However, reports are still inadequate and it will be interesting to evaluate the introduction of the reporting model in future years.Clin Chem Lab Med 2007;45:254–60.

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by degeneration of the anterior horn cells of the spinal cord, causing symmetric proximal muscle weakness. SMA is classified in three clinical types, SMA I, SMA II, and SMA III, based on the severity of the symptoms and the age of onset. About 95% of SMA cases are caused by homozygous deletion of the survival motor neuron 1 (SMN1) gene (5q13), or its conversion to SMN2. The molecular diagnosis of this disease is usually carried out by a polymerase chain reaction-restriction fragment length polymorphism approach able to evidence the absence of both SMN1 copies. However, this approach is not able to identify heterozygous healthy carriers, which show a very high frequency in general population (1:50). We used the multiple ligation-dependent probe amplification (MLPA) approach for the molecular diagnosis of SMA in 19 affected patient and in 57 individuals at risk to become healthy carriers. This analysis detected the absence of the homozygous SMN1 in all the investigated cases, and allowed to discriminate between SMN1 deletion and conversion to SMN2 on the basis of the size showed by the peaks specific for the different genes mapped within the SMA critical region. Moreover, MLPA analysis evidenced a condition of the absence of the heterozygous SMN1 in 33 out of the 57 relatives of the affected patients, demonstrating the usefulness of this approach in the identification of healthy carriers. Thus, the MLPA technique represents an easy, low cost, and high throughput system in the molecular diagnosis of SMA, both in affected patients and in healthy carriers.

This study describes the largest series reported to date, of individuals belonging to unrelated families carrying a beta-thalassaemia-like phenotype in whom the beta-globin gene was found to be structurally intact by sequence analysis. This genetic determinant appears haematologically heterogeneous, displaying either a silent beta-thalassaemia-like phenotype or a typical beta-thalassaemia carrier-like phenotype in different families. Compound heterozygosity for both beta-thalassaemia-like determinant and typical beta-thalassaemia allele resulted either in thalassaemia intermedia or thalassaemia major. By linkage analysis both the silent and the typical beta-like determinants were found not to be linked to the beta-globin cluster. Sequence analysis of the hypersensitive site cores of locus control region and of the genes coding for the transcription factors erythroid Kruppel-like factor and nuclear factor (erythroid-derived 2) were normal. beta-globin mRNA levels determined by real-time polymerase chain reaction were reduced in both types of beta-like carriers. These results indicate the existence of causative genetic determinants not yet molecularly defined, but most likely, resulting from either the reduction or loss of function of a gene coding for unknown transcriptional regulator(s) of the beta-globin gene. The knowledge of these rare beta-thalassaemia-like determinants have implications for clinical and, especially, prenatal diagnosis of beta-thalassaemia.

Summary Background Pemphigus foliaceus (PF) is an autoimmune blistering disease of unknown aetiology, which is endemic in Brazil. Although the pathogenesis of PF is still unknown, proteins of the contact system have been implicated. Objectives As the components of the kinin system may interact with those of the contact system, in this study we evaluated the plasma levels of high-molecular-weight kininogen (HK) and low-molecular-weight kininogen (LK), and the activity of plasma kallikrein, tissue kallikrein and kininase II in plasma of patients with PF presenting with Nikolsky's sign. As kidneys and salivary glands are relevant sources of tissue kallikrein for plasma, we also evaluated urinary/salivary kallikrein and urinary kininase II activities. Methods Fifteen patients and 15 age- and sex-matched controls were studied. Kininogen levels were determined by enzyme-linked immunosorbent assay, and the activities of kallikreins and kininase II were determined using selective chromogenic substrates. Results Compared with controls, plasma HK levels were decreased (P = 0·031), whereas the activities of plasma kallikrein, tissue kallikrein and kininase II in plasma, and the activity of salivary kallikrein, were increased in patients (P

Autoimmune endocrinopathies are characterised by an increased number of peripheral blood lymphocytes (PBL) expressing activation/ memory markers on their surface. The aim of this study was to determine whether a similar finding could be detected in a group of 11 paediatric and young adult patients suffering from autoimmune polyglandular syndrome type 1 (APS1), also called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), as very few data have previously been reported in this field. The control group was made up of 11 sex- and age-matched healthy subjects. Fifteen lymphocyte subsets were compared, in terms of percentage and absolute number, and statistical analysis was performed by the Mann-Whitney test. Measurement of T (CD3+), B (CD19+), natural killer (NK, CD3-CD16/56+), CD4+ and CD8+ T lymphocytes showed that patients with APS1 had a higher percentage and absolute count of T lymphocytes: this was entirely due to the statistically larger CD3+CD4+ fraction. Patients with APS1 also had slightly fewer B and NK lymphocytes, but the difference was negligible. Comparison of CD4+ subpopulations bearing activation and naive/memory antigens (marked by CD69, CD25, anti-HLA-DR, CD45RA and CD45RO) showed that patients with APS1 had generally larger percentages and absolute counts of these subsets: however, only the percentage and absolute size of the CD4+CD25+ subset (p = 0.0354 and p = 0.0151, respectively), and the absolute number of the CD4+ anti-HLA-DR+ and CD4+ CD45RO+ subsets (p = 0.0193 and p = 0.0209, respectively) were significantly higher. Interestingly, patients with APS1 also had significantly fewer CD8+CD11b+ and CD3-CD8+ cells. In conclusion, PBL distribution in APS1 resembles that of other autoimmune diseases. Further studies are needed to confirm and possibly extend these data.

To report the experiences on preimplantation genetic diagnosis (PGD) in couples at risk for beta-thalassaemia in Sardinia.23 couples at risk for beta-thalassaemia were included in the PGD programme with a total of 42 cycles performed. Among these, 11 couples were fertile, while the remaining 12 had associated fertility problems. In vitro Fertilization (IVF), PGD and prenatal genetic molecular confirmation protocols and results are reported.All the patients followed the protocol of ovarian stimulation, oocyte retrieval, intracytoplasmic sperm injection (ICSI), embryo biopsy and genetic analysis. A total of 272 oocytes were fertilized in the regular way, and embryo biopsy was performed on 202 embryos. Out of these 202 embryos, 192 (95%) were successful. The genetic diagnosis was performed on 150 embryos (78.1%). Ninety-eight were identified as unaffected and 75 were transferred in 31 cycles. In the infertile patient group, two biochemical pregnancies (11.1% per transfer), in the fertile patient group, four clinical pregnancies, two twin and two singleton pregnancies (30.8% per transfer), were obtained. The genetic molecular results were confirmed in all pregnancies by first-trimester chorionic villus sampling (CVS).Our study shows that PGD for beta-thalassaemia is an available procedure for couples who wish to avoid termination of pregnancy, except in cases where the IVF cycle efficiency is very poor.

The silent beta-thalassemia mutation, beta(+)-101C--T, is the only mutation currently described in the distal beta-globin CACCC box. We present a novel mutation, a C--G transversion, in the same position. Expression analysis in heterozygous subjects demonstrated that the mutation determines a 20% reduction in the output of the beta-globin gene. DNA-protein interaction and transactivation analysis correlated the decrease in the beta-globin synthesis with the reduced binding and transactivation of EKLF to the mutant promoter. These data predict that the beta-101C--G mutation will display a silent thalassemia phenotype similar to that of the beta-101C--T mutation.

Summary Stem-cell transplantation can cure β thalassaemia. We aimed to assess whether fetal HLA typing done early in the pregnancy of couples who were at risk of β thalassaemia could provide an alternative to pregnancy termination if the prospect of a bone-marrow transplantation from a family member was available. In our clinic in Sardinia, we did fetal HLA typing for 49 couples at risk of having a baby with β thalassaemia. Two affected children were born and successfully received a transplantation from a family donor. Five non-affected fetuses were HLA compatible with an affected sibling and their cord blood was harvested for a future transplantation.

In this study, we have defined by molecular analysis, the alpha, beta, and delta globin genotype in a group of individuals with normal or thal-like red cell indices but borderline hemoglobin (Hb)A2 levels, who were identified in a program for beta-thal carrier screening. In 37 of 125 individuals with borderline HbA2 levels, we detected a molecular defect in the beta, in both the delta and the beta, or in the alpha globin gene. Specifically seven of these subjects were carriers of the -101 C T mutation, ten of the IVSI nt6 T C mutation, 16 were double heterozygotes for delta and beta thal, and two had the triple alpha globin gene and two the single alpha globin gene deletion. From these results, we may conclude that subjects with borderline HbA2, particularly when they marry a typical beta-thal carrier, should be extensively investigated in order not to miss heterozygous beta-thalassemia.

Programs of prospective carrier screening and genetic counseling for beta-thalassemia among couples planning marriage, preconception, or during early pregnancy are ongoing in several at-risk populations in the Mediterranean area, including Greeks, Greek Cypriots and Continental Italians. Carrier detection is carried out by haematological analysis followed by mutation detection by DNA analysis. Once carrier couples are identified, prenatal diagnosis is accomplished by mutation analysis on PCR amplified DNA from chorionic villi. These programs have been very effective, due to education programs and subsequent acceptance of screening. Future prospects include automation of the process of mutation detection by microchips analysis, introduction of preconception and preimplantation diagnosis and hopefully fetal diagnosis by analysis of fetal cells in maternal circulation.

In this study, we have carried out molecular analysis of the AIRE (autoimmune regulator) gene in 11 patients (from 8 families) affected by autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, originating from a restricted area of Southern Italy (the Salento peninsula in Puglia). Of the 16 mutant AIRE alleles from the 8 probands studied, 12 carried a missense mutation (W78R in 9, P539L in 2, and P252L in 1), 2 carried the Q358X nonsense mutation, and 2 carried the 1058delT frameshift mutation. All these mutations except the 1058delT are novel. Each of the detected mutations either predicts a premature termination of the protein or results in a nonconservative amino acid change, most likely adversely affecting the function of the protein. The W78R missense mutation is relatively common in these patients, having been detected (in homozygosity or compound heterozygosity) in 6 of the 8 probands tested, indicating the presence of a founder effect. The results of this study contribute to the delineation of the molecular pathology of the AIRE gene and enhance our ability to perform a molecular diagnosis in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy patients from Southern Italy.

Cardiovascular diseases are the first cause of death in our Country. They mainly manifests in adult age but it is the result of initiated lesions since the young age and imputable often to errors of behaviours and to non appropriate styles of life. The knowledges related to the prevention of some illnesses, allows a reduction of the incidence of these, a reduction of the mortality, with consequent reduction of the health and social costs related to the care and to the rehabilitation. In our educational system, unlike what happens in the most greater part of the other European countries, these themes are only partially present and however treated in sporadic and insufficient way. For these raisons Pronto Cuore onlus Association has decided to start, in collaboration with the Regione Lazio, a project of health education to the high schools students considering that a more informed population has a longer expectancy of life and a better life quality. This job wants to underline the necessity to undertake a health education program to teach and inform students and teachers: to recognize some factors of risk as principal causes of cardiovascular diseases; to change life style; to recognize critical situations and behaviours to be adopted.