An improved method for the diagnostic approach of alpha+-thalassaemia.

An improved method for the diagnostic approach of alpha(+)-thalassaemia is described. The method is based on five common parameters: absence of iron deficiency, mild morphological abnormalities of erythrocytes, normal or slightly reduced erythrocytic indices MCV and MCH, normal chromatographic findings, and presence of haemoglobin H inclusions in erythrocytes with methyl-violet stain after, but not before, incubation with oxidant agent. We studied by DNA analysis, 58 subjects fulfilling the above mentioned diagnostic criteria and we found that 50 of them (86.2%) had a alpha-globin gene defect. In the remaining eight subjects (13.8%) no alpha-gene defect could be documented with the techniques used in the DNA analysis, which detect the six well-known alpha(+)-thalassaemic defects in the Greek population. We conclude that the improved method, we described has a high sensitivity and accuracy in the screening of alpha(+)-thalassaemia.

Evaluation of the BeTha gene 1 kit for the qualitative detection of the eight most common Mediterranean beta-thalassemia mutations.

We describe the evaluation of the Bio-Rad BeTha Gene 1 kit (Bio-Rad Laboratories, Hercules, CA), a DNA-probe assay designed for the qualitative determination of the eight most common Mediterranean beta-thalassemia mutations. The kit utilizes the principle of allele-specific oligonucleotide (ASO) hybridization. Following sample preparation and in vitro DNA amplification by the polymerase chain reaction (PCR), an allele-specific detection of the amplified products by a nonradioactive enzymatic assay is performed. Genomic DNA is prepared from an individual's whole blood with a DNA purification matrix. In a second step, the beta-globin gene is amplified in a multiplex PCR reaction containing four 5' biotinylated oligonucleotide primers. In a final step, an aliquot of the PCR reaction is first chemically denatured and then captured in two eight-well strips of a 96-well enzyme-linked immunosorbent assay (ELISA) plate by hybridization to an immobilized ASO probe. Each DNA sequence at each of the eight mutation sites is represented by one normal and one mutant ASO. During this capture/hybridization step, which is performed at 37 degrees C, only perfectly matched PCR products will be captured by an ASO. Subsequently, the allele-specific captured biotin-labeled PCR products are detected by a colorimetric enzymatic reaction. The system permits the detection of 16 beta-thalassemia alleles using a high-throughput format that can be automated easily. A clinical feasibility study was performed to evaluate the functionality (method comparison study, assay validity using samples previously collected and stored at various temperatures for different periods of time, interference on kit performance, and assay validity for prenatal diagnosis) and the usability (ease of use, sample throughput) of the kit. The analysis of 110 samples previously studied with reference methods showed 100% clinical sensitivity and specificity. We demonstrate here that the procedure not only increases the throughput of beta-thalassemia allele genotyping but also provides an accurate, rapid, reliable, and nonisotopic diagnostic tool.

Prenatal diagnosis of X-linked spinal and bulbar muscular atrophy in a Greek family.

X-linked spinal and bulbar muscular atrophy (SBMA) is a late-onset motor neuron disorder which is caused by an expansion of the trinucleotide repeat (CAG)n in the first exon of the androgen receptor gene. Two cases of prenatal testing for the disease in a Greek family are reported. An affected male died in his late 50s of this disorder and his 30-year-old daughter (an obligate carrier) asked for prenatal testing for SBMA. DNA analysis revealed that she indeed carried an expanded allele of 40 repeats, as well as a normal size allele of 24 repeats. Prenatal diagnosis of SBMA was performed when, on two successive pregnancies, two male fetuses with the expanded (CAG)n allele were found.

Beta-thalassaemia mutations and the underlying beta gene cluster haplotypes in the Greek population.

The polymorphic sites across the beta gene cluster (restriction haplotypes) in association with specific thalassaemic mutations were analyzed in representative samples of normal and thalassaemic Greeks in comparison to similar data of other populations around the mediterranean basin. We studied 316 normal chromosomes, 218 chromosomes from patients with thalassaemia major and 72 chromosomes from patients with thalassaemia intermedia. In the former group, haplotype frequencies followed the order I, IX, II, V etc.. In the group of patients with transfusion-dependent thalassaemia the order was I, II, V and VI, while in those with thalassaemia intermedia the most frequent haplotypes were I and VI. The frequency of haplotypes I and VI was higher among the thalassaemic chromosomes in comparison to those of the normal population; haplotype IX showed the inverse relation. These findings imply that the thalassaemic mutations occurred at a very early stage on haplotypes I and VI and much later on haplotype IX. Micromapping did not reveal any significant variations. Haplotypes I, II, V and VI were associated with the molecular defects IVS-1 nt 110, beta zero-39, IVS-1 nt 1 and IVS-1 nt 6 respectively. A number of other mutations were also identified. The molecular defect was identified also on a random sample of beta-thalassaemia carriers (424 chromosomes). On the basis of the overall data, the feasibility of prenatal diagnosis of thalassaemia by allele specific hybridization is ca. 80%, with the four most common oligomers and 95% when the set of probes expands to eight.

The origin of the sickle mutation in Greece; evidence from beta S globin gene cluster polymorphisms.

Study of the Hpa I polymorphism 3' to the beta-globin gene in the Greek population revealed absence of the site in 238 beta S chromosomes, in contrast to a much larger sample of chromosomes carrying the beta A gene, where this site was consistently positive. Subsequent haplotype analysis of the beta-globin gene cluster in 82 beta S chromosomes demonstrated that 79 (96%) belonged to haplotype #19, while the three exceptions (all Hpa I negative) could be explained by a delta-beta recombination event. Haplotype #19 was never encountered in a parallel study of the 83 beta A chromosomes. Comparison of the above results with similar surveys in other parts of the world and consideration of various historical events suggest that the beta S mutation was introduced into Greece over the last few centuries by the Saracen raids and/or by settlements of North African slaves brought in by the Arabs, Franks, Venetians, or Ottoman Turks, who have occupied the country over the last millennium.

Selective survival of only the healthy fetus following prenatal diagnosis of thalassaemia major in binovular twin gestation.

Selective feticide is the procedure of choice when, in twin binovular pregnancy, only one of the fetuses is shown to be affected. As the probabilities for this condition are almost 1:2 when the genetic disease is due to homozygosity for two autosomal recessive genes, the problem is expected to occur frequently among the ever increasing number of couples seeking prenatal diagnosis of thalassaemia and the haemoglobinopathies. The present report is the first case of this condition and the ninth in the overall medical literature.

Structural study of hemoglobin Knossos, beta 27 (B9) Ala leads to Ser. A new abnormal hemoglobin present as a silent beta-thalassemia.

A new electrophoretically silent hemoglobin variant is described that produces the classical phenotype of beta thalassemic intermedia in association with beta thalassemia trait. This variant has the expression of a silent beta thalassemia trait. The abnormal hemoglobin was detected by acid-urea-Triton-acrylamide electrophoresis and further demonstrated by isoelectric focusing. The amount of the variant in carrier is approximately 30% of the total hemoglobin. No instability was found. Absence of hemoglobin A in the propositus blood facilitated structural studies. Peptides maps were normal but analysis of individual peptide spots showed an Ala leads to Ser substitution in the beta T3. This variant has been previously called Hb Knossos (beta 27 (B9) Ala leads to Ser).