Two novel truncating variants of the AAAS gene causative of the triple A syndrome.

PURPOSE: 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. METHODS: Genetic analysis of AAAS gene in triple A syndrome patient and molecular and functional characterization of the novel identified allelic variants. RESULTS: 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. CONCLUSIONS: 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.

Short communication: novel truncating mutations in the CFTR gene causing a severe form of cystic fibrosis in Italian patients.

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.

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.

Two novel mutations of the AIRE protein affecting its homodimerization properties.

We report two novel mutations, c.230T>C (p.F77S) and c.64_69del (p.V22_D23del) within the HSR domain of the AIRE protein in two patients of Italian descent affected by APECED. Both mutations were found in the compound heterozygous state respectively with c.994+5G>T and c.232T>A (p.W78R). With the two-hybrid assay in the yeast system we found that constructs containing the two mutations fail to interact with the wild-type protein. These findings indicate that both mutations negatively affected the homodimerization properties of the AIRE protein, thereby leading to a defective function.

[Significance of health education in schools. Strategy for the prevention of cardiovascular diseases]. / L'importanza dell'educazione sanitaria nelle scuole. Una strategia per la prevenzione delle malattie cardiovascolari.

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.

A beta-thalassaemia allele with 3 base substitution in codons 4/5 & 6 (ACT CCT GAG-> ACA TCT TAG) detected by denaturing gradient gel electrophoresis & sequencing.

We report the analysis of a beta-thalassaemia gene involving three bases in codons 4/5 and 6 (ACT CCT GAG-> ACA TCT TAG) in a confirmed carrier whose child had beta-thalassaemia major. The fragment of the gene carrying the mutation was detected by denaturing gradient gel electrophoresis (DGGE) using GC clamped primers, followed by direct sequencing. DGGE analysis indicated that one gene was the wild type (normal) while the sequence changes observed were all in the other gene causing beta-thalassaemia major in the child. This confirms a single case report from Lucknow (UP) and adds to the beta-thalassaemia mutations identified in the beta-globin gene in India and will help in the thalassaemia control programme.

High-risk pregnancy in beta-thalassemia major women. Report of three cases.

Reproductive failure is common in beta-thalassemia major patients because of endocrine damage resulting from iron overload. Here 3 full-term pregnancies following spontaneous ovulation in 2 splenectomized beta-thalassemia major women are reported. The main echocardiographic parameters, such as left ventricular end-diastolic and end-systolic diameters, fractional shortening and ejection fraction, were within the normal range before pregnancy, but worsened during gestation, and 1 patient developed pre-congestive heart failure. Deferoxamine therapy was continued throughout 2 pregnancies, while in the other it was stopped after 8 weeks: no abnormalities were noted in the children. Thanks to the currently applied therapies, an increased number of pregnancies may now be expected in beta-thalassemia major women: it is important to find out more about the pregnancy-related problems and their management in these patients.

Prenatal Diagnosis and Screening of the Haemoglobinopathies.

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.

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

Autoimmune polyendocrinopathy-candidiasisectodermal 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.

Prenatal diagnosis and screening of the haemoglobinopathies.

This paper reviews the most important aspects of carrier detection procedures, genetic counselling, population screening and prenatal diagnosis of the thalassaemias and sickle cell anaemia. Carrier detection can be made retrospectively, following the birth of an affected child, or prospectively. Carrier detection and genetic counselling in at-risk populations for alpha-thalassaemia and sickle cell anaemia is carried out mostly retrospectively. However prospective carrier screening is ongoing in Cuba and Guadeloupe for sickle cell anaemia and, in a very limited way, in some South East Asian populations, for alpha-thalassaemia. For beta-thalassaemia, several programmes, based on carrier screening and counselling of couples at marriage, preconception or early pregnancy, are operating in several Mediterranean at-risk populations. These programmes have been very effective, as indicated by increasing knowledge on thalassaemia and its prevention by the target population and by the marked decline of 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 maternal circulation.

Molecular diagnosis and carrier screening for beta thalassemia.

Thalassemias are common autosomal recessive disorders especially in populations of Mediterranean, Middle Eastern, and Far Eastern descent. Relatively high incidence is also observed in people of Asian Indian origin but the incidence is more limited in those of African descent. Beta Thalassemias are heterogeneous at the molecular level, with more than 150 different molecular defects identified to date. Despite this heterogeneity, each at-risk population has its own spectrum of common mutations, usually from 5 to 10, a finding that simplifies mutation analysis. Homozygosity for beta thalassemias usually results in transfusion-dependent thalassemia major and, rarely, in mild non-transfusion-dependent conditions. Molecular diagnosis may be used to define genotypes associated with mild forms. Advances in carrier diagnosis using hematologic analysis followed by mutation analysis have made possible the population screening of women at childbearing age and prenatal diagnosis. This approach in combination with nondirective genetic counseling has resulted in a consistent decline of the birth of affected homozygotes in several Mediterranean at-risk populations, as well as knowledge of the risks of being a carrier. Molecular diagnosis of homozygotes and identification of carriers of beta thalassemia may lead to improved clinical management of patients with the disorder and prevention of the birth of affected homozygotes.

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.

HB Hinwil or beta 38(C4)Thr-->Asn: a new beta chain variant detected in a Swiss family.

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.

Control of beta-thalassaemia by carrier screening, genetic counselling and prenatal diagnosis: the Sardinian experience.

Homozygous beta-thalassaemia in a number of at-risk populations (Greek and Turkish Cypriots, Greeks, Continental Italians and Sardinians) has been prevented at the population level by programmes based on carrier screening, genetic counselling and prenatal diagnosis. The Sardinian experience is based on a 20-year programme. Voluntary screening has been offered to prospective parents and, primarily, to women with an ongoing pregnancy. Education of the population at large, training of health personnel, and use of posters and informative booklets have been critical elements for the success of the programme. Genetic counselling has been carried out in a non-directive manner following well-established guidelines. The use of extended family screening magnified the efficacy of the screening programme, allowing the identification of the large majority of parents at risk by screening only 13% of the population at child-bearing age. Following counselling, the large majority of parents accepted prenatal diagnosis. Definition of the parents' mutation and prenatal diagnosis were carried out by a number of PCR-based procedures. The programme was effective, as indicated by the reduction of the birth rate of thalassaemia major from 1:250 live births to 1:4000.

A specific cystic fibrosis mutation (T3381) associated with the phenotype of isolated hypotonic dehydration.

We carried out molecular screening for mutations in the cystic fibrosis transmembrane regulator (CFTR) gene in eight children of Sardinian descent seen because of hypotonic dehydration associated with hyponatremia, hypochloremia, hypokalemia, and metabolic alkalosis; none had pulmonary or pancreatic involvement. All the patients had the T3381 mutation either in homozygosity or compound heterozygosity with another CF mutation. The T3381 mutation was not detected in patients with CF who had classic symptoms or in healthy persons of the same descent. These data suggest that the T3381 mutation is associated with a specific mild CF phenotype.

A promoter mutation, C-->T at position -92, leading to silent beta-thalassaemia.

This study describes the clinical phenotype of the C-->T mutation at position -92 of the beta-globin gene. Excluding two cases with HbA2 levels within the range of the beta-thalassemia carrier state, heterozygotes for this mutation showed normal or borderline red blood cells count, Hb levels, MCV, MCH and HbA2 values, and unbalanced globin chain synthesis. Compound heterozygotes for the -92 C-->T mutation and a beta zero-thalassaemia mutation (beta zero 39) (two cases) or severe beta+-thalassaemia (beta+ IVSII nt 745) (two cases) developed thalassaemia intermedia. According to these characteristics, the -92 promoter mutation should be added to the list of silent beta-thalassaemias.