M T Scalas

PRENATAL DIAGNOSIS OF BETA-THALASSAEMIA WITH THE SYNTHETIC-OLIGOMER TECHNIQUE

103 couples attending the antenatal clinic in Sardinia were screened for the beta o-39 (nonsense) mutation, which codes for beta-thalassaemia, with the oligonucleotide method. In 94 couples both members had the beta o-39 mutant and thus were eligible for antenatal testing with this method. These pregnancies were monitored with amniocentesis (61) or trophoblast biopsy (33). Prenatal diagnosis in those monitored with amniocentesis was carried out with DNA analysis of uncultivated amniocytes (19) or cultivated cells (38). In 4 pregnancies results were unsatisfactory, and prenatal diagnosis was repeated with fetal-blood analysis. Trophoblast biopsy was unsuccessful in 1 pregnancy and gave a misdiagnosis in another because of maternal contamination. In the latter case the genotype of the fetus was established with amniocyte DNA analysis and globin-chain-synthesis studies.

Beta thalassaemia mutations in Sardinians: implications for prenatal diagnosis.

In this study we have characterised by oligonucleotide hybridisation and direct restriction endonuclease analysis the beta thalassaemia mutation in 494 Sardinian beta thalassaemia heterozygotes. The most prevalent mutation, accounting for 95.4% of the cases, was the nonsense mutation at codon 39. The remainder, in decreasing order of frequency, were a frameshift at codon 6 (2.2%), beta + IVS-1, nt 110 (0.4%), and beta + IVS-2, nt 745 (0.4%). This information allows prenatal diagnosis by DNA analysis to be made in the great majority of Sardinian couples at risk for beta thalassaemia.

Prenatal diagnosis of beta thalassaemia by oligonucleotide analysis in Mediterranean populations.

We have used four oligonucleotide probes and two restriction enzymes to detect the beta thalassaemia mutation in a group of 61 couples of Italian descent who were prospective parents. We have been able to define the beta thalassaemia mutation in both parents in 47 couples and in only one parent in 12 couples. Prenatal diagnosis was accomplished successfully either by amniocyte (two) or trophoblast (26) DNA analysis in 28 couples in which the pregnancy was in progress. These results indicate that direct identification of the mutation by oligonucleotide or restriction endonuclease analysis is a practical and useful method for prenatal diagnosis of beta thalassaemia in childless couples.

Reliability of prenatal diagnosis of genetic diseases by analysis of amplified trophoblast DNA.

Dot blot analysis on enzymatically amplified trophoblast DNA with allele specific oligonucleotide probes is currently used for the prenatal diagnosis of single gene disorders characterised at the molecular level, such as the beta thalassaemias, phenylketonuria, sickle cell anaemia, and alpha 1-anti-trypsin deficiency. A potential problem with the use of this procedure is the co-amplification of maternal sequences, which may obscure the diagnosis in the fetus. To address this question, we carried out prenatal diagnosis of beta thalassaemia in 300 couples at risk by dot blot analysis on enzymatically amplified DNA with 32P or horseradish peroxidase labelled allele specific oligonucleotide probes. We verified the diagnosis obtained by this procedure with oligonucleotide hybridisation on electrophoretically separated non-amplified trophoblast DNA fragments. We detected no co-amplified maternal sequences, even with a faint signal, in the dot blot of trophoblast DNA from those fetuses diagnosed as normal or homozygotes, nor in those diagnosed as heterozygotes, who were born to parents carrying different mutations and had inherited the paternal mutation. These results indicate that, when careful dissection of trophoblast tissue from maternal decidua is carried out, amplification of chorionic villi DNA is not associated with amplification of maternal DNA sequences. We may thus conclude that dot blot analysis of trophoblast DNA is a very reliable procedure for prenatal diagnosis.

Prenatal Diagnosis of β-Thalassemia by the Analysis of Trophoblast DNA with the Synthetic Oligomer Technique

Trophoblast DNA has been recently used for the antenatal diagnosis of inherited hemoglobinopathies (Williamson et al. 1981; Old et al. 1982; Goossens et al. 1983). The main advantage of this method is that chorionic villi may be obtained at an early stage of gestation, which may make prenatal diagnosis a more acceptable procedure for an increasing number of couples at risk.