Alessandra Meloni

A common mutation in Sardinian autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy patients

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED; also called APS-1,) is a rare autosomal recessive disorder that is more frequent in certain isolated populations. It is characterized by two of the three major clinical symptoms that may be present: Addison’s disease, and/or hypoparathyroidism and/or chronic mucocutaneous candidiasis. We have recently identified the gene for APECED, which we termed AIRE (for autoimmune regulator). AIRE is expressed in thymus, lymph nodes and fetal liver, and encodes a protein with two putative zinc fingers and other motifs suggestive of a transcriptional regulator. Seven mutations have been described to date, including R257X, the predominant Finnish and northern Italian APECED allele, which has also been observed in other patients of diverse origin on different haplotypes. A 13-bp deletion (1094–1106del) has also been observed in several patients of different geo-ethnic origin. The other described mutations appear to be rare. We present mutational analyses of the AIRE gene in ten Sardinian APECED families and show that there is a mutation, R139X, associated with one predominant haplotype unique to the Sardinian patients (18/20 independent alleles). The carrier frequency of R139X in Sardinia is 1.7%, giving an estimated population frequency of APECED of 1/14,400. Using linkage disequilibrium data, the estimated age of the R139X mutation is between 20 and 25 generations. A previously described 13-bp deletion was also observed on an allele of one patient. The identification of a single common Sardinian APECED mutation will facilitate its genetic diagnosis. Given the carrier frequency of R139X in the Sardinian population, AIRE may be implicated in the pathogenesis of other autoimmune diseases in the Sardinian population, particularly those affecting the endocrine system.

Molecular screening and fetal diagnosis of β-thalassemia in the Italian population

SummaryThis paper reports our experience of molecular screening and fetal diagnosis of β-thalassemia in 457 at risk couples of Italian descent. Molecular screening was carried out by dot blot analysis on amplified DNA with oligonucleotide probes complementary to the eight most common mutations in Italians [β∘39 (C→T); ∘6 (-A); β+-87 (C→G); β+ IVSI nt 110 (G→A); β∘IVSI nt 1 (G→A); β+ IVSI nt 6 (T→C); β∘ IVSII nt 1 (G→A); β+ IVSII nt 745 (C→G)]. By using this approach, we have been able to define the mutation in 92.8% of cases. The rest (all but four) were defined by direct sequencing and this led to the detection of nine rare mutations [β∘76 (-C); β+ IVSI nt 5 (G→A); β+ IVSI nt 5 (G→C); β+ IVSI -1 (cod 30) (G→C); β+-87 (C→T), β∘-290 bp del.; β+-101 (C→T)], and to the characterization of a novel mutation consisting of the deletion of the G at the invariant AG of the IVSII splice acceptor site of the β-globin gene (β IVSII nt 850-1 bp). In the remaining four cases, the β-globin gene showed entirely normal sequences and the β-globin gene cluster was intact, as indicated by Southern blot analysis. Fetal diagnosis was carried out by dot blot analysis with the oligonucleotide probes defined in the parents. The procedure is simple and reliable, and the results can be obtained within 1 week of sampling. No misdiagnosis has so far occurred. The results indicate that fetal diagnosis of β-thalassemia by DNA analysis may be obtained in practically all cases (even in a population showing marked heterogeneity of β-thalassemia) by the combination of dot blot analysis for detecting common mutations, and direct sequencing for defining those that are uncommon.

Overexpression of the Cytokine BAFF and Autoimmunity Risk

BACKGROUND Genomewide association studies of autoimmune diseases have mapped hundreds of susceptibility regions in the genome. However, only for a few association signals has the causal gene been identified, and for even fewer have the causal variant and underlying mechanism been defined. Coincident associations of DNA variants affecting both the risk of autoimmune disease and quantitative immune variables provide an informative route to explore disease mechanisms and drug‐targetable pathways. METHODS Using case–control samples from Sardinia, Italy, we performed a genomewide association study in multiple sclerosis followed by TNFSF13B locus–specific association testing in systemic lupus erythematosus (SLE). Extensive phenotyping of quantitative immune variables, sequence‐based fine mapping, cross‐population and cross‐phenotype analyses, and gene‐expression studies were used to identify the causal variant and elucidate its mechanism of action. Signatures of positive selection were also investigated. RESULTS A variant in TNFSF13B, encoding the cytokine and drug target B‐cell activating factor (BAFF), was associated with multiple sclerosis as well as SLE. The disease‐risk allele was also associated with up‐regulated humoral immunity through increased levels of soluble BAFF, B lymphocytes, and immunoglobulins. The causal variant was identified: an insertion–deletion variant, GCTGT→A (in which A is the risk allele), yielded a shorter transcript that escaped microRNA inhibition and increased production of soluble BAFF, which in turn up‐regulated humoral immunity. Population genetic signatures indicated that this autoimmunity variant has been evolutionarily advantageous, most likely by augmenting resistance to malaria. CONCLUSIONS A TNFSF13B variant was associated with multiple sclerosis and SLE, and its effects were clarified at the population, cellular, and molecular levels. (Funded by the Italian Foundation for Multiple Sclerosis and others.)

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.

Promoter mutations producing mild β‐thalassaemia in the Italian population

Summary. In this study we have investigated the molecular basis for a mild form of β‐thalassaemia in three patients of Italian descent. In two, belonging to different families and affected by a mild and late‐presenting form of thalassaemia major, direct sequencing of amplified DNA detected a C→T substitution at position −87 of the β‐globin gene in the compound heterozygous state either with codon 39 nonsense mutation or β+IVSI, nt 110 mutation. The −87 (C→T) mutation has been previously described, in combination with the β+IVSI, nt 110 mutation, in a single patient with thalassaemia intermedia. Both our patients showed a more severe phenotype as compared to that resulting from compound heterozygosity for a severe β‐thalassaemia mutation and another promoter mutation (−87, C→G) at the same position. In the third patient with the thalassaemia intermedia phenotype, we detected a novel promoter mutation, consisting in a C→A substitution at position −86, in combination with the codon 39 nonsense mutation. The results of this study indicate that different nucleotide substitutions affecting the proximal CACCC box of the β‐globin gene in combination with severe β‐thalassaemia, produce a mild form of thalassaemia ranging in severity from thalassaemia intermedia to late‐presenting thalassaemia major.

Differential secretion of the mutated protein is a major component affecting phenotypic severity in CRLF1-associated disorders

Crisponi syndrome (CS) and cold-induced sweating syndrome type 1 (CISS1) are disorders caused by mutations in CRLF1. The two syndromes share clinical characteristics, such as dysmorphic features, muscle contractions, scoliosis and cold-induced sweating, with CS patients showing a severe clinical course in infancy involving hyperthermia, associated with death in most cases in the first years of life. To evaluate a potential genotype/phenotype correlation and whether CS and CISS1 represent two allelic diseases or manifestations at different ages of the same disorder, we carried out a detailed clinical analysis of 19 patients carrying mutations in CRLF1. We studied the functional significance of the mutations found in CRLF1, providing evidence that phenotypic severity of the two disorders mainly depends on altered kinetics of secretion of the mutated CRLF1 protein. On the basis of these findings, we believe that the two syndromes, CS and CISS1, represent manifestations of the same disorder, with different degrees of severity. We suggest renaming the two genetic entities CS and CISS1 with the broader term of Sohar–Crisponi syndrome.

The forkhead transcription factor Foxl2 is sumoylated in both human and mouse: sumoylation affects its stability, localization, and activity.

The FOXL2 forkhead transcription factor is expressed in ovarian granulosa cells, and mutated FOXL2 causes the blepharophimosis, ptosis and epicanthus inversus syndrome (BPES) and predisposes to premature ovarian failure. Inactivation of Foxl2 in mice demonstrated its indispensability for female gonadal sex determination and ovary development and revealed its antagonism of Sox9, the effector of male testis development. To help to define the regulatory activities of FOXL2, we looked for interacting proteins. Based on yeast two-hybrid screening, we found that FOXL2 interacts with PIAS1 and UBC9, both parts of the sumoylation machinery. We showed that human FOXL2 is sumoylated in transfected cell lines, and that endogenous mouse Foxl2 is comparably sumoylated. This modification changes its cellular localization, stability and transcriptional activity. It is intriguing that similar sumoylation and regulatory consequences have also been reported for SOX9, the male counterpart of FOXL2 in somatic gonadal tissues.

Molecular analysis of patients of Sardinian descent with Crigler-Najjar syndrome type I.

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.

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.

Crigler-Najjar syndrome type II resulting from three different mutations in the bilirubin uridine 5′-diphosphate-glucuronosyltransferase (UGT1A1) gene

Editor—Crigler-Najjar syndromes (CN, MIM 218800) are inborn errors of metabolism characterised by unconjugated hyperbilirubinaemia resulting from the defective activity of the hepatic enzyme bilirubin uridine 5′-diphosphate-glucuronosyltransferase (B-UGT). CN syndrome has been classified into two types according to the degree of hyperbilirubinaemia and to the response to phenobarbital administration. The more severe CN type I is characterised by severe chronic non-haemolytic unconjugated hyperbilirubinaemia with high levels of serum bilirubin owing to the absence of bilirubin UGT activity. In the milder CN type II, bilirubin UGT activity is only decreased and a consistently significant reduction is obtained with phenobarbital treatment, which does not occur in CN I. Like other members of the UGT isozyme family, the two human liver bilirubin UGT isozymes, UGT1A1 and UGT1D, are encoded by the UGT1 gene complex through a mechanism of alternative splicing. Each gene has a unique promoter and a unique exon 1, while exons 2-5 are common to both genes.1 Most of the enzymatic activity results from the expression of the UGT1A1 gene.2 At the molecular level, CN I results from a number of different defects; nonsense (or frameshift) and missense mutations are represented in almost the same amounts both in homozygosity and in the compound heterozygous state.3 4 The milder phenotype in CN II patients seems to be mainly the result of homozygosity for missense mutations5 and more …