Hb Las Palmas or alpha 2 beta 2(49)(CD8)Ser----Phe, a mildly unstable hemoglobin variant

A new hemoglobin variant with a Ser----Phe substitution at position beta 49(CD8) was discovered in two members of a family living in the Canary Islands, Spain. Detection was by polyacrylamide gel electrophoresis and by reversed phase high performance liquid chromatography. The variant, which constituted 43% and 45%, respectively, in the two heterozygotes, was slightly unstable. Its presence did not affect hematological values though there was a mild reticulocytosis.     

Beta-thalassemia intermedia in two Turkish families is caused by the interaction of Hb Knossos [beta 27(B9)Ala----Ser] and of Hb City of Hope [beta 69(E13)Gly----ser] with beta (0)-thalassemia

We have studied a few members of two Turkish families, who had a beta-thalassemia of the intermediate type. An abnormal hemoglobin was found in both families, which when present in association with beta(0)-thalassemia was considered to be the primary cause for the increased severity of the disease. In the first family this variant was Hb Knossos [beta 27(B9)Ala----Ser] which occurred together with the frameshift in codon #8 type of beta(0)-thalassemia. This compound heterozygosity, observed for the first time in the Turkish population was characterized by a considerable increase in Hb F production, mainly of the G gamma type, as expected for a chromosome with haplotype IV. In the second family, the variant was Hb City of Hope [beta 69(E13)Gly----Ser] which was present in combination with an unknown type of beta-thalassemia. The increase in Hb F production in the compound heterozygote was minimal. Reversed phase high performance liquid chromatography and the DNA amplification-synthetic oligonucleotide probe procedure were major tools in identifying the different abnormalities.     

Newer developments in the identification of beta-thalassemia

One of the easiest and most sensitive methods of detecting mutations in the beta-globin gene leading to beta-thalassemia is by the use of oligonucleotide probes. The current method involves digestion of 5-10 micrograms of genomic DNA followed by gel electrophoresis, and blotting onto nitrocellulose. The membrane is then hybridized with a 32P-radiolabeled oligonucleotide probe containing the specific point mutation of interest. Finally, the membrane is subjected to X-ray film for 3-10 days. We wish to report a method for detecting these mutations which involves 1 microgram of genome DNA or less. The method involves the use of a gene amplification technique. A series of primers are synthesized which span the beta-globin gene. In each primer set, one primer is complementary to the beta-gene and the other primer is complementary to the non-coding strand. The suspected mutation point is located between these two primers. With the use of this primer set, the beta-globin gene region is amplified by denaturing, annealing, and DNA synthesis. The amplification cycle is repeated 25 to 30 times. The amplification is conducted using the Klenow fragment of DNA polymerase I or Taq polymerase in the presence of all four deoxynucleotide triphosphates. The resulting amplified DNA is applied to a nylon membrane with the aid of a dot-blot apparatus and directly hybridized with normal and mutant deoxynucleotide probes. The entire process requires one to two days. More than 300 beta-thalassemia homozygotes have been identified in our laboratories; over 20 different mutations have been observed.     

Validation of a differential PCR and an ELISA procedure in studying HER-2/neu status in breast cancer

HER-2/neu oncogene status and total cellular p185^HER-2 content were simultaneously analyzed in 415 invasive breast-cancer specimens by differential PCR and ELISA respectively. Mathematical analysis of the data led us to establish a cut-off value of 1.7 for the ratio between the intensity of the HER-2/neu gene band and the reference gene band, to consider the HER-2/neu gene amplified, and of 260 fmol/mg protein, to consider p185^HER-2 over-expressed. Of the 415 tumors studied, 15% showed a diverse degree of HER-2/neu gene amplification. Of these tumors, 87% showed over-expression of the p185^HER-2. Of the remaining 352 specimens that did not display HER-2/neu gene amplification, 97% showed no p185^HER-2 over-expression (p < 0.0001). In 40 selected samples with a p185^HER-2 level lower than 260 fmol/mg protein, the degree of p185^HER-2 phosphorylation was very low or undetectable. Conversely, 38 of 46 selected tumors with a p185^HER-2 level higher than 260 fmol/mg protein exhibited a considerable degree of p185^HER-2 phosphorylation (p < 0.0001). Our data suggest that: (i) differential PCR and ELISA, which are relatively simple procedures, give similar information on HER-2/neu status in breast cancer; and (ii) given the large series analyzed, the cutoff values established can be considered as safe values for determining whether, in a given tumor, the HER-2/neu oncogene is amplified or p185^HER-2 is over-expressed. © 1996 Wiley-Liss, Inc.     

Two new large deletions resulting in epsilon gamma delta beta-thalassemia

Detailed gene mapping data are provided for members of a Yugoslavian and Canadian family with a thalassemia heterozygosity characterized by mild anemia with severe microcytosis and hypochromia, normal levels of Hb A2 and slightly raised Hb F levels. The condition in both families results from large deletions (minimally approximately 148 kb in the Yugoslavian family and minimally approximately 185 kb in the Canadian family), which include all functional and psi genes of the beta globin gene cluster. The Canadian propositus was a newborn baby who has been followed for nearly 2 years; severe anemia developed some 30-40 days after birth when the Hb F level was still 70%; recovery was evident at the age of 90 days when the Hb F level had decreased to 40%.     

Mild and severe beta-thalassemia among homozygotes from Turkey: identification of the types by hybridization of amplified DNA with synthetic probes

Through the procedure of gene amplification combined with hybridization to synthetic 19 base pair (bp) oligonucleotide probes, it has been possible to identify nine different mutations in the DNA of 47 subjects from Turkey and Northern Cyprus with a beta-thalassemia homozygosity. The IVS-I nucleotide (nt) 110 G----A and the IVS-I nt 6 T----C substitutions and the frameshift at codon 8 were most frequently observed. Direct correlations were made between these data and clinical observations; mild disease was associated with homozygosity for IVS-I nt 6 T----C, for frameshift at codon 8, for the C----G substitution at nt -87, and for IVS-I nt 5 G----T, and for a double heterozygosity for some of these conditions. Moderate disease, observed in some of the patients, could be explained by combinations of specific mutations. All mutations were associated with specific haplotypes, while in some the observed beta zero-thalassemia was of the mild type due to a considerable production of Hb F.     

Characterization of a newly discovered α-thalassaemia-1 in two Spanish patients with Hb H disease

A new deletion of more than 27 kb, removing the psi zeta 1, psi alpha 2, psi alpha 1, alpha 2, alpha 1 and theta 1 globin genes has been found in four members of a Spanish family, including two patients with Hb H disease. The 5' end point of the deletion is located between the zeta and psi zeta genes, and the 3' end of the deletion is downstream of the 3' hypervariable region.