Valeria Faà

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.

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.

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.

A promoter mutation, C → T at position ‐92, leading to silent /3‐thalassaemia

Summary. This study describes the clinical phenotype of the C?→? T mutation at position – 92 of the β‐globin gene. Excluding two cases with HbA2 levels within the range of the /3‐thalassaemia 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 β° thalassaemia mutation (β°39) (two cases) or severe β‐thalassaemia (p+ IVSII nt 745) (two cases) developed thalassaemia intermedia. According to these characteristics, the –92 promoter mutation should be added to the list of silent β‐thalassaemias.

Thalassaemia-like carriers not linked to the β-globin gene cluster

This study describes the largest series reported to date, of individuals belonging to unrelated families carrying a β‐thalassaemia‐like phenotype in whom the β‐globin gene was found to be structurally intact by sequence analysis. This genetic determinant appears haematologically heterogeneous, displaying either a silent β‐thalassaemia‐like phenotype or a typical β‐thalassaemia carrier‐like phenotype in different families. Compound heterozygosity for both β‐thalassaemia‐like determinant and typical β‐thalassaemia allele resulted either in thalassaemia intermedia or thalassaemia major. By linkage analysis both the silent and the typical β‐like determinants were found not to be linked to the β‐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. β‐globin mRNA levels determined by real‐time polymerase chain reaction were reduced in both types of β‐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 β‐globin gene. The knowledge of these rare β‐thalassaemia‐like determinants have implications for clinical and, especially, prenatal diagnosis of β‐thalassaemia.

Preconceptional identification of cystic fibrosis carriers in the Sardinian population: A pilot screening program

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%).

β-defensin CNV is not associated with susceptibility to Candida albicans infections in Sardinian APS I patients.

OBJECTIVE The 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 pre-disposition 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 one-way ANOVA 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 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.