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sults indicate that fetal diagnosis of B-thalassemia by DNA analysis may .... studied. Po delta. Campania n: 1a. 762. Puglia. Calabria Basilicata Sicilia. Othersu ...
Ifum Genet

(1992) 89:585-589

O

Springer-Verlag 1992

Review article

Molecular screening and fetal diagnosis of B-thalassemia in the Italian population Maria Cristina Rosatellit, Teresa Tuveril, Maria'feresa Scalasl, Giovan llattista Leoni2, Raftraela Sardul, Valeria Faà1, Alessandra Meloni3, N1aria Alessanclra Pischccldal, Maria Demurtas2, Giovanni Monni?, and Antonio Caol llstituto di Clinica e Biologia dell'Età Evolutiva, Università dcgli Studi, Vizr Jenner s/n, I-09100 Cagliari, Italv 2Ospedale Regionale per le Nlicrocitemie, USL 21., Cagliari, Italy 3Istituto di Ricerca sulle Talassemie e Anemie N{editerranee. CNR, Cagliari, Italy Received November 22,1997 / Revised Janaury 22, 1992

Summary. This paper reports our experience of molecular screening and fetal diagnosis of B-thaiassernia 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 [p"39 (C-+T); p"6 (-A); P*-87 (C-+G); []+ MI nt 110 (G--+1i); B"IVSI nt 1 (G--+A); B+IVSI nt 6 (T--.>C); B"IVSII nt 1 (G=A)t 0+IVSII nt "/45 (C-=G)]. By using this approach, we have been able to define the mutation in92.8% of cases. The rest (a11 but four) rvere defined by direct sequencing and this led to the detection of nine rare mutations IB'76

(-C); P+IVSI nt 5 (G--+A); F+IVSI nt 5 (G--+C); B+ IVSI -1 (cod 30) (G-+C); P*-87 (C--+T), B'-290bp dei.; B+ -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 Bglobin gene ([] IVSII nt 850 -1bp). in the remaining four cases, the B-globin gene showed entirely normal sequences and the B-globin gene cluster was intact, as indicated by Southern blot analysis. Fetal diagnosis was carried out by dot blot anaiysis with the oligonucleotide probes defined in the parents. The procedure is simple and reliable, and the resuits can be obtained within 1 week of sampling. No misdiagnosis has so far occurred. The results indicate that fetal diagnosis of B-thalassemia by DNA analysis may be obtained in practically all cases (evep iri a population showing marked heterogeneity of B-thalassemia) by the combination of dot blot analysis for detecting common inutations, and direct sequencing for defining those that are uncommon.

lntroduction Fetal diagnosis of B-thalassemia is nowadays carried out by a number of different procedures on enzymatically Correspondence to:

A. Cao

amplified trophoblast DNA. Known mutations can be detected by al1e1ic specific oligonucleotide probes (Ristaldi et al. 1989), primer specific oligonucleotides (Newton et al. 1989) or restriction endonuclease analysis (Pirastu et al. 1989). Unknown mutations are defined by denaturiirg gradient gel electrophoresis (Fisher and Lerman 1983;M,vers et al. 1985), single strand conformation polymorphism analysis (Orita et al. 1989) or direct sequencing (Church and Gilbert 1984). The main problem encountered so far in fetal diagnosis of B-thalassemia is the marked molecular heterogeneity of this disorder, causing difficulties in the practical organization of fetal testing. In this paper, we report our experience of molecular screening and fetai diagnosis of B-thalassemia by trophoblast DNA analysis in the Italian population, which may be considered an example of a popuÌation with marked molecular heterogeneity o[ the p-thalussemias.

Subjects Childless couples @: a5i) of Italian descent at risk for B-thalassemia, referred to our Genetic Service either for counselling and/ or prenatal diagnosis, are the subjects of this investigation.

Methods Three segments of the p-globin gene were enzymatically amplified by the polymerase chain reaction (PCR) (Saiki et al. 1988) according to the primers given in Fig.1. We used 25 ampiifying cycles.

The definition of the B-thalassemia mutation within the first segment was obtained in prospective parents by the analysis of either one or other of the amplified products; prenatal diagnosis was carried out by analyzing both products. The B-thalassemia mutations were defined by dot blot analysis on amplified DNA with either 32P or horseradish-peroxidase-labelied oligonucleotide probes complementary to the eight most common mutations in the Italian population, viz. B"39 (C+T); B"6 (-A); 0+-87 (C+G); P+ IVSI nt 110 (G+A); B'IVSI nt 1 (G+ A); 0+ NSI nt 6 (T+C); P" IVSII nt 1 (G+A); B+ IVSII nt 745

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blot analysis was used to delìne those mutations resulting frorn gross structural rearrangemerrt. Any mutatioll eluding definition by this approach rverc characterized by direct sequencing of tire amplified single strand DNA, according to the dideoxy-chain termination method of San-

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