Alpha-thalassaemia prenatal diagnosis by two PCR-based methods.

In Cyprus all couples carrying alpha0-thalassaemia mutations are detected in the course of the thalassaemia carrier screening program and prenatal diagnosis is offered to all of them. Prenatal diagnosis for alpha-thalassaemia is routinely done by two independent molecular methods. With the first method, the mutations of the parents are directly determined by gap-PCR and then the chorionic villus sample (CVS) is examined for the presence of these mutations. With the other method, a (CA)n repeat polymorphic site located between the psialpha1- and alpha2-globin genes is used for determining the presence or absence of the normal and mutant alleles. In the period from 1995 to 1999, molecular analysis of 46 couples in which haematological data were consistent with deletion of two alpha-globin genes in both partners indicated that only 13 of them were actually at risk for haemoglobin (Hb) Bart's hydrops fetalis and prenatal diagnosis was provided in 16 pregnancies. The molecular diagnosis was possible in all cases with the use of both gap-PCR and (CA)n repeat polymorphisms analysis. No misdiagnosed cases for alpha-thalassaemia have been reported to date.

Hb Bart's levels in cord blood and alpha-thalassemia mutations in Cyprus.

The purpose of this study was to examine the frequency of alpha-thalassemia in the population of Cyprus using cord blood samples. The levels of Hb Bart's were compared with the hematological indices and the results correlated with the presence of alpha-thalassemia mutations. The protocols for the polymerase chain reaction detection of the six most common alpha-globin mutations encountered in Cyprus were optimized, and the frequency of each mutation was determined through the screening of 495 random cord blood samples. The total allele frequency for the mutations examined was 10.6%, of which 1% is due to the triplication of the alpha-globin genes. The -alpha(3.7 kb) deletion accounts for 72.8% of all detectable mutations, while the--MED-I and -(alpha)-20.5 kb mutations account for 7.8%. The level of Hb Bart's and the MCV and MCH values in cord blood samples were found to correlate closely with the severity of alpha-thalassemia, although the -alpha(3.7 kb) deletion and perhaps other mild alpha-thalassemia mutations may not give detectable Hb Bart's levels. A reasonably accurate estimate of the alpha-thalassemia carrier frequency may be obtained from cord blood studies if Hb Bart's estimates are combined with hematological indices. When molecular methods are added, these give the best way to use cord bloods to survey populations for alpha-thalassemia.

Comparative in vivo expression of beta(+)-thalassemia alleles.

Double heterozygotes who inherit one abnormal though stable beta-globin variant in association with a molecularly identified beta(+)-thalassaemia allele provide unique opportunities to quantify the in vivo expression of particular beta(+)-thalassemia alleles. The globin products of the two alleles can be separated, quantified and the output of the beta(+)-thalassaemia allele expressed as the MCH-beta(A) in pg beta(A)-globin/beta(+)-thalassemia allele/RBC = 0.5 MCH x Hb A%. In this communication we provide new quantitative data on the expression of five mutations as follows: the beta(+)-87 (C-->G) = 3.8 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 1); the beta(+) IVS-I-1 (G-->A) = 0.2 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 1); the beta(+) IVS-I-6 (T-->C) = 2.9 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 7); the beta(+) IVS-I-110 (G-->A) = 1.1 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 13), and the beta(+) IVS-II-745 (C-->G) = 1.74 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 2). The values obtained are compared with those of other beta(+)-thalassemia alleles from the literature. It can be seen that the MCH-beta(A) value may be a correct index of thalassemia severity useful for the correlation of genotype with phenotype, and for understanding the effects of mutations in beta-globin genes on pathophysiologically meaningful beta-globin gene expression.

alpha-Thalassaemia in the population of Cyprus.

We have determined the alpha-thalassaemia (alpha-thal) determinants in 78 patients with Hb H disease from Cyprus; 25 were Turkish Cypriots and 53 were Greek Cypriots. Four deletional and three non-deletional alpha-thal alleles were present; the -alpha(3.7 kb) alpha-thal-2 and the --MED-I alpha-thal-1 were most frequently seen; --MED-II and -(alpha)20.5 deletions occurred at considerably lower frequencies. About 15% of all chromosomes carried a non-deletional alpha-thal-2 allele; of these the 5 nucleotide (nt) deletion at the first intervening sequence (IVS-I) donor splice site was present in approximately 8% of all chromosomes. Two types of polyadenylation signal (poly A) mutations were observed. No striking frequency differences were seen between Greek and Turkish Cypriot patients. Combinations of the various types of alpha-thal resulted in eight different forms of Hb H disease. The phenotypes were comparable except for great variations in the level of Hb H which was highest (average approximately 22%) in the 12 patients with the alpha 5nt alpha/--MED-I combination. One patient with the same form of Hb H disease but with an additional beta-thal (IVS-I-110,G-->A) heterozygosity had a most severe microcytosis and hypochromia with < 1% Hb H. Variations in the level of Hb H might correlate with the severity of the disease, although this was not evident from the haematological data.

Analysis of delta-globin gene mutations in Greek cypriots.

We recently described four delta-globin gene mutations in Greek Cypriots studied by polymerase chain reaction (PCR) amplification and automated fluorescence-based DNA sequence analysis (Blood 78:3298, 1991). Selective restriction enzyme digestion of PCR products facilitated direct mutation detection. Twenty-eight additional samples from unrelated Cypriots with Hb A2 levels ranging from 0.6% to 3.6% were studied by PCR and showed the following: twelve had the delta 27 (ala-->ser) mutation, one was heterozygous for the delta IVS-2 AG-->GG change, and none had either the delta 116 (arg-->cys) or delta 141 (leu-->pro) mutations. The remaining samples were divided into two groups: 11 with borderline normal Hb A2 values that were not pursued; and four with abnormal Hb A2 values. The delta-globin genes from these four samples were sequenced and the same four changes identified in each: a C-->T at -199, a C-->T at codon 4 (thr-->ile), a silent C-->T at codon 97, and an AT deletion at position 722 in IVS-2. The codon 4 change abolishes a Ple I site whereas the codon 97 creates an Nla III site, thus facilitating rapid identification. All four changes are in cis position, suggesting that the -199 C-->T, the C-->T at codon 97, and the AT deletion in IVS-2 are neutral polymorphisms present on the codon 4 (thr-->ile) chromosome. DNA haplotype analysis suggests all five delta-globin gene mutant alleles arose independently on different chromosomal backgrounds.