A simple electrophoretic procedure for fetal diagnosis of beta-thalassaemia due to short deletions.

This study describes three couples at risk for homozygous beta-thalassaemia in which one of the partners carried a short deletion beta-thalassaemia defect. Detection of short deletions in trophoblast DNA was accomplished by the very simple procedure of non-denaturing polyacrylamide gel electrophoresis. This method may be applied to detect beta-thalassaemia mutations due to deletion or addition of more than two nucleotides.

Molecular screening and fetal diagnosis of beta-thalassemia in the Italian population.

This paper reports our experience of molecular screening and fetal diagnosis of beta-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 [beta zero 39 (C----T); beta zero 6 (-A); beta+ -87 (C----G); beta+ IVSI nt 110 (G----A); beta zero IVSI nt 1 (G----A); beta+ IVSI nt 6 (T----C); beta zero IVSII nt 1 (G----A); beta+ 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 [beta zero 76 (-C); beta+ IVSI nt 5 (G----A); beta+ IVSI nt 5 (G----C); beta+ IVSI -1 (cod 30) (G----C); beta+ -87 (C----T), beta zero -290 bp del.; beta+ -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 beta-globin gene (beta IVSII nt 850 -1 bp). In the remaining four cases, the beta-globin gene showed entirely normal sequences and the beta-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 beta-thalassemia by DNA analysis may be obtained in practically all cases (even in a population showing marked heterogeneity of beta-thalassemia) by the combination of dot blot analysis for detecting common mutations, and direct sequencing for defining those that are uncommon.

Prenatal diagnosis of inherited hemoglobinopathies.

This paper reviews the techniques presently available for the prenatal diagnosis of inherited hemoglobinopathies. At the present time, mutations of the globin genes are detected directly in trophoblast DNA, enzymatically amplified by polymerase chain reaction. Known mutations may be defined by restriction endonuclease digestion, non denaturing gradient gel electrophoresis, allele specific oligonucleotide probes or allele specific oligonucleotide primers. Unknown mutations are detected by denaturing gradient gel electrophoresis followed by direct sequencing. Other potentially useful methods for unknown mutations are single strand conformation polymorphism analysis and chemical mismatch cleavage analysis. A potential pitfall for all procedures based on analysis of amplified DNA is the coamplification of maternal sequences. This may be avoided by a careful dissection of maternal decidua from fetal trophoblast, by using an amount of chorionic villi not inferior to 5-10 mg and by reducing the number of amplifying cycles to approximately 20. Monitoring the presence of co-amplified maternal sequences by the analysis of polymorphic sequences is strongly recommended. Future perspectives consist of preimplantation diagnosis by biopsy of the morula or blastula or ova genotyping by analysis of the second polar body.

Promoter mutations producing mild beta-thalassaemia in the Italian population.

In this study we have investigated the molecular basis for a mild form of beta-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 beta-globin gene in the compound heterozygous state either with codon 39 nonsense mutation or beta +IVSI, nt 110 mutation. The -87 (C----T) mutation has been previously described, in combination with the beta +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 beta-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 beta-globin gene in combination with severe beta-thalassaemia, produce a mild form of thalassaemia ranging in severity from thalassaemia intermedia to late-presenting thalassaemia major.

Molecular characterization of beta-thalassemia in the Sardinian population.

This study reports the molecular characterization of beta-thalassemia in the Sardinian population. Three thousand beta-thalassemia chromosomes from prospective parents presenting at the genetic service were initially analyzed by dot blot analysis with oligonucleotide probes complementary to the most common beta-thalassemia mutations in the Mediterranean at-risk populations. the mutations which remained uncharacterized by this approach were defined by denaturing gradient gel electrophoresis (DGGE) followed by direct sequence analysis on amplified DNA. We reconfirmed that the predominant mutation in the Sardinian population is the codon 39 nonsense mutation, which accounts for 95.7% of the beta-thalassemia chromosomes. The other two relatively common mutations are frameshifts at codon 6 (2.1%) and at codon 76 (0.7%), relatively uncommon in other Mediterranean-origin populations. In this study we have detected a novel beta-thalassemia mutation, i.e., a frameshift at codon 1, in three beta-thalassemia chromosomes. The DGGE procedure followed by direct sequencing on amplified DNA is a powerful approach for the characterization of unknown mutations in this genetic system. The results herein presented allowed an expansion of the applicability of prenatal diagnosis by DNA analysis, to all couples at risk for beta-thalassemia in our population.

Molecular bases for cystic fibrosis in the Sardinian population.

Cystic fibrosis (CF) is a relatively uncommon genetic disorder in the Sardinian population. In this study, we have defined the frequency of the most common CF mutation (delta F508) and carried out a genotype-phenotype correlation analysis in a group of 21 patients with CF and of Sardinian descent. We detected the delta F508 mutation in 24 (57%) out of the 42 CF chromosome investigated. This mutation was found in the homozygous state in 9 patients and in the heterozygous state in 6 patients. The remaining 6 patients had other mutations. The delta F508 mutation was associated only with the KM19/XV2c 2 1 haplotype. Genotype-phenotype correlation analysis did not give clear-cut results, probably because of the small number of patients investigated. However, out of the four patients with meconium ileus, three were homozygous and one was heterozygous for the delta F508 mutation, confirming that the presence of delta F508 or other severe mutations in the homozygous state is the prerequisite for the development of meconium ileus.

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.