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.

Correct human ␤-globin mRNA has been restored in erythroid cells from transgenic mice carrying the human gene with ␤-globin IVS2-654 splice mutation and from thalassemia patients with the IVS2-654/␤ E genotype. This was accomplished in a dose-and time-dependent manner by free uptake of morpholino oligonucleotide antisense to the aberrant splice site at position 652 of intron 2 in ␤-globin pre-mRNA. Under optimal conditions of oligonucleotide uptake, the maximal levels of correct human ␤-globin mRNA and hemoglobin A in patients' erythroid cells were 77 and 54%, respectively. These levels of correction were equal to, if not higher than, those obtained by syringe loading of the oligonucleotide into the cells. Comparison of splicing correction results with the cellular uptake of fluorescein-labeled oligonucleotide indicated that the levels of mRNA and hemoglobin A correlate well with the nuclear localization of the oligonucleotide and the degree of erythroid differentiation of cultured cells. Similar but not as pronounced results were obtained after the oligonucleotide treatment of bone marrow cells from IVS2-654 mouse. The effectiveness of the free antisense morpholino oligonucleotide in restoration of correct splicing of IVS2-654 pre-mRNA in cultured erythropoietic cells from transgenic mice and thalassemic patients suggests the applicability of this or similar compounds in in vivo experiments and possibly in treatment of thalassemia.

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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.

The activity of human paraoxonase 2 (PON2) is rapidly reduced in cells incubated with the bacterial quorormone 3-Oxo-dodecanoyl Homoserine Lactone (3OC12HSL), an observation that led to hypothesize a fast PON2 posttranslational modification (PTM). Recently, we detected a 3OC12HSL-induced PTM in a cell-free system in which a crude extract from 3OC12HSL-treated HeLa cells was able to inactivate and ubiquitinate at position 144 a recombinant PON2. Here we show the occurrence of this and new PTMs on PON2 in HeLa cells. PTMs were found to gather nearby the two SNPs, A148G, and S311C, that are related to type-2 diabetes and its complications. Furthermore, we detected a PTM nearby a 12 amino acids region that is deleted in PON2 Isoform 2. An in vitro mutation analysis showed that the SNPs and the deletion are involved in PON2 activity and suggested a role of PTMs on its modulation, while a SAXS analysis pointed to Isoform 2 as being largely unstructured, compared to the wild type. Besides, we discovered a control of PON2 expression via a putative mRNA operon involving the Wilms tumor 1 associated protein (WTAP) and the E3 ubiquitin ligase (E3UbL) baculoviral IAP repeat-containing 3 (BIRC3).

LETTERS TO THE EDITORHb Foggia or ␣ ␣117(GH5)Phe→ →Ser: a new ␣ ␣2 globin allele affecting the ␣ ␣Hb-AHSP interaction We report a novel ␣ ␣2-globin gene allele with the mutation cod 117 TTC>TCC or ␣ ␣117(GH5)Phe>Ser detected in three carriers with ␣ ␣-thalassemia phenotype. The mutated mRNA was present in the reticulocytes in the same amount as the normal one, but no chain or hemoglobin variant were detected. Most likely the amino acid substitution impairs the interaction of the ␣ ␣-chain variant with the AHSP and prevents its stabilizing effect, thus leading to the ␣ ␣-chain pool reduction.

We characterized mutations and haplotypes of the delta-globin gene (HBD, MIM# 142000) in two regions of southern Italy. Mutations were discovered by screening for individuals with Hb A2<2%. In Basilicata, about 10,000 students were screened and 53 carriers in 43 unrelated families were diagnosed; in Campania, cases were referred through a routine thalassemia counseling service. Twelve alleles were detected. Four were novel variants [Hb A2-Metaponto (g.238C>A), Hb A2-Campania (g.302C>A), Hb A2-Lucania (g.393C>G), and Hb A2-Capri (g.443G>T)]. Hb A2-Lucania was not inherited but had arisen in the propositus. Two were novel mutations in the noncoding regions: the substitutions IVS2+6T>A, presumably affecting the splicing, and g.-126A>T in the GATA motif presumably affecting transcription. All novel alleles were found associated with haplotypes common in the Mediterranean area. The remaining six were alleles already described. The Hb A2-Yialousa (g.82G>T) was the most prevalent (42/63 families). Recurrent homologous crossing-over events have, most likely, linked this allele to Haplotypes IX (24 families), IV (10 families), or III (seven families). The ratio of Haplotypes IX:IV:III was about the same in the two regions. The rare allele Hb A2-NYU (g.39T>A) was found in 11 families from Basilicata associated with Haplotype I. All the 11 families lived in a restricted area extending from the Ionian Coast for 15 km along the Angri and Sinni Rivers. A founder effect most probably gave origin to this isolated group. The remaining four alleles were rare: the 7.2-kb deletion Corfù type (HBD g.-5946_1262del), Hb A2-Mitsero (g.14C>T), Hb A2-Etolia (g.385T>C), Hb A2-Coburg (g.1376G>A). Correlation between genotype and phenotype was established in 103 carriers.