Mononuclear cells from peripheral blood of thalassemic patients were treated with morpholino oligonucleotides antisense to aberrant splice sites in mutant ␤-globin precursor mRNAs (premRNAs). The oligonucleotides restored correct splicing and translation of ␤-globin mRNA, increasing the hemoglobin (Hb) A synthesis in erythroid cells from patients with IVS2-654͞␤ E , IVS2-745͞ IVS2-745, and IVS2-745͞IVS2-1 genotypes. The maximal Hb A level for repaired IVS2-745 mutation was Ϸ30% of normal; Hb A was still detectable 9 days after a single treatment with oligonucleotide. Thus, expression of defective ␤-globin genes was repaired and significant level of Hb A was restored in a cell population that would be targeted in clinical applications of this approach. O ne of the most common genetic diseases of mankind is ␤-thalassemia. It affects large populations in the Mediterranean basin, Middle East, South East Asia, and Africa. Approximately 80 million people are carriers of the thalassemia trait and the percentage of carriers worldwide is increasing. Concomitant increases in the number of patients, presently numbering several hundred thousands, are held down by high infant mortality in underdeveloped countries, by population screening, genetic counseling, and abortions; the growing need for clinical treatment is evident (1).The disease is due to mutations causing defective ␤-globin gene expression and deficiency of ␤-globin and adult hemoglobin (Hb) A. Homozygotes or compound heterozygotes for severe defects are affected with thalassemia major or Cooley's anemia, lethal if untreated, and suffer pronounced anemia, bone deformities, and hepatomegaly and splenomegaly (2). Regular, lifelong transfusions combined with iron chelation constitute current treatment. Bone marrow transplantation, the only cure, is limited by the scarcity of suitable donors and facilities. Experimental protocols to stimulate synthesis of fetal hemoglobin by sodium butyrate or hydroxyurea (3-6) or ␤-globin gene repair or replacement (4,7,8) have not yet been fully tested at the clinical level. Clearly there is a need for alternative treatments to replace the costly and cumbersome transfusion regimen.More than 100 thalassemic mutations causing defective ␤-globin gene expression and ␤-globin deficiency have been identified, but the ones causing aberrant splicing are among the most common (9). They are found in intron 1 (IVS1-5, IVS1-6, and IVS1-110) and intron 2 (IVS2-654, IVS2-705, and IVS2-745) of the ␤-globin gene (9-15). A mutation in codon 26 of the gene also results in activation of an aberrant splicing pathway and a mutated ␤-globin protein (␤ E ) (16,17). A common pathogenic feature of these mutations is activation of aberrant splice sites and modification of the splicing pathways, even though the correct splice sites remain potentially functional.Aberrant splicing of several of the ␤-globin splicing mutants has been shown in cell free extracts (18), in transfected HeLa based cell lines (19-22), and in a transgenic mouse model (23).More importan...

SummaryA family from the Southeast of Italy was found to have a novel b-globin mutant, b+45 G fi C, with the features of a silent b-thalassaemia mutation. It was asymptomatic in two heterozygotes, but its interaction with the severe thalassaemia mutation b-IVS-II-654 C fi T worsened the haematological and biosynthetic phenotype in two compound heterozygotes; moreover, another compound heterozygote, who was also heterozygote for the aaa anti3AE7 , suffered from thalassaemia intermedia. The mutation was found associated in cis with the IVS-II-754 T fi C substitution, which did not lead to abnormally spliced mRNA. Furthermore, the amount of b+45 mRNA was the same as the bA mRNA in the reticulocytes of the carriers. In vitro transcription/translation experiments demonstrated that the b+45 G fi C decreased the efficiency of translation of the b-globin chain by about 30%: this slight impairment was consistent with the observed clinical phenotype. The b+45 G fi C is the first mutation found in the Kozak sequence (GACACCATGG) of the b-globin gene and the first one at the position -6 upstream the ATG. The Kozak consensus sequence plays a major role in the initiation of translation process. The present finding supports the hypothesis that the G in position -6 is important in this process.

The alpha-globin chains are encoded by two duplicated genes (HBA2 and HBA1, 5'-3') showing overall sequence homology >96% and average CG content >60%. alpha-Thalassemia, the most prevalent worldwide autosomal recessive disorder, is a hereditary anemia caused by sequence variations of these genes in about 25% of carriers. We evaluated the overall sensitivity and suitability of DHPLC and DG-DGGE in scanning both the alpha-globin genes by carrying out a retrospective analysis of 19 variant alleles in 29 genotypes. The HBA2 alleles c.1A>G, c.79G>A, and c.281T>G, and the HBA1 allele c.475C>A were new. Three pathogenic sequence variations were associated in cis with nonpathogenic variations in all families studied; they were the HBA2 variation c.2T>C associated with c.-24C>G, and the HBA2 variations c.391G>C and c.427T>C, both associated with c.565G>A. We set up original experimental conditions for DHPLC and DG-DGGE and analyzed 10 normal subjects, 46 heterozygotes, seven homozygotes, seven compound heterozygotes, and six compound heterozygotes for a hybrid gene. Both the methodologies gave reproducible results and no false-positive was detected. DHPLC showed 100% sensitivity and DG-DGGE nearly 90%. About 100% of the sequence from the cap site to the polyA addition site could be scanned by DHPLC, about 87% by DG-DGGE. It is noteworthy that the three most common pathogenic sequence variations (HBA2 alleles c.2T>C, c.95+2_95+6del, and c.523A>G) were unambiguously detected by both the methodologies. Genotype diagnosis must be confirmed with PCR sequencing of single amplicons or with an allele-specific method. This study can be helpful for scanning genes with high CG content and offers a model suitable for duplicated genes with high homology.