T. P. Molchanova

Access to a Syllabus of Human Hemoglobin Variants (1996) Via the World Wide Web

Information on mutations in human hemoglobin is important in many efforts, including understanding the pathophysiology of hemoglobin diseases, developing therapies, elucidating the dynamics of sequence alterations inhuman populations, and dissecting the details of protein structure/function relationships. Currently, information is available on a large number of mutations and variants, but is distributed among thousands of papers. In an effort to organize this voluminous data set, two Syllabi have been prepared compiling succinct information on human hemoglobin abnormalities. In both of these, each entry provides amino acid and/or DNA sequence alterations, hematological and clinical data, methodology used for characterization, ethnic distribution, and functional properties and stability of the hemoglobin, together with appropriate literature references. A Syllabus of Human Hemoglobin Variants (1996) describes 693 abnormal hemoglobins resulting from alterations in the alpha-, beta-, gamma-, and delta-globin chains, including special abnormalities such as double mutations, hybrid chains, elongated chains, deletions, and insertions. We have converted this resource to an electronic form that is accessible via the World Wide Web at the Globin Gene Server (http://globin.cse.psu.edu). Hyperlinks are provided from each entry in the tables of variants to the corresponding full description. In addition, a simple query interface allows the user to find all entries containing a designated word or phrase. We are in the process of converting A Syllabus of Thalassemia Mutations (1997) to a similar electronic format.

The differences in quantities of alpha 2-and alpha 1-globin gene variants in heterozygotes

Summary. We have identified through sequencing of amplified DNA the mutations in the α2‐ and α1‐globin genes in 63 individuals with a heterozygosity for an α chain abnormal haemoglobin (Hb). Moreover, we developed a reverse transcription/polymerase chain reaction (RT/PCR) based procedure for the determination of the α2‐ and α1‐ mRNA ratio in normal individuals. The numbers of α2 and α1 variants were nearly the same. The average precentage of the abnormal Hb in heterozygotes with α2 mutations (23.5%) was slightly higher than that in heterozygotes with α mutations (19.7%) (stable Hbs only). These percentages correspond to a ratio of α2 to α1 of 1.19 to 1 at the protein level. Variations in the number of active α‐globin genes and in the stability of the variants (greatly) affected the percentages of the abnormal protein. The average ratio between the α2‐ and α1‐mRNAs in 12 normal individuals was 2.6–2.75 to 1, about as expected from published data. and 2.0 to 1 for two persons with an α‐thalassaemia‐2 (α‐thal‐2) (‐3.7 kb) heterozygosity. The high relative mRNA (α2) level which is about twice the relative level of the α2 protein suggests a less efficient translation of the α2‐mRNA.

A simplified procedure for sequencing amplified DNA containing the α2- or α1-Globin gene

(1994). A simplified procedure for sequencing amplified DNA containing the α2- or α1-Globin gene. Hemoglobin: Vol. 18, No. 3, pp. 251-255.

Hb Alesha or alpha 2 beta (2)67(E11) Val->Met : a new unstable hemoglobin variant identified through sequencing of amplified DNA

We have identified a valine-->methionine mutation at position 67 of the beta chain in the hemoglobin of a young Russian patient with severe hemolytic disease, anemia, splenomegaly, Heinz body formation, and continued requirement for blood transfusions despite an early splenectomy. Sequencing of amplified DNA readily identified a GTG-->ATG mutation at codon 67. The introduction of the larger methionine residue into the heme pocket, and the loss of the bonds between valine at beta 67 and the heme group, adequately account for the severe instability of Hb Alesha and the serious clinical condition of its carrier.

A Second Observation of the Fetal Methehoglobin Variant HB F-M-Fort Ripley or α2Gγ292(F8)HIS→TYR

We have identified a second baby with the fetal methemoglobin F-M-Fort Ripley. It was observed in a Caucasian infant from Canada; at least eleven additional members of that family were known to have had a neonatal cyanosis similar to that seen in the propositus and in a previously described baby (2). Sequencing of amplified DNA that included (part of) the Gγ gene greatly facilitated the characterization. The Gγx chain was readily isolated by reversed phase high performance liquid chromatography; its quantity was ∼12.5% of total γ. Interestingly, the baby also carried the AγT mutation on one chromosome, either in cis or in trans to the Gγx mutation. Hb F-M-Fort Ripley could be isolated in reasonably pure form by DEAE-cellulose chromatography. The isolated Hb Fx was unstable, had spectral changes characteristic for the M-hemoglobins, while its methemqglobin derivative reacted rapidly with cyanide. Oxygen affinity data could not be obtained. It is suggested that the formation of a rather large amount (∼25%) ...

Hb Hradec Kralove (Hb HK) or alpha 2 beta 2 115(G17)Ala->Asp, a severely unstable hemoglobin variant resulting in a dominant beta-thalassemia trait in a Czech family

We have identified through sequencing of amplified DNA a GCC-->GAC mutation in codon 115 of the beta-globin gene in a mother and daughter of a small Czech family. This base change was confirmed by hybridization with a 32P-labeled specific oligonucleotide probe and by gene mapping because it creates a new Ava II site. The mutation results in an Ala-->Asp replacement at beta 115(G17); this beta chain is severely unstable and could not be identified either as chain or as hemoglobin variant by isoelectrofocusing and various high performance liquid chromatography methods. Stability tests were mildly positive in freshly prepared lysates, but an unstable hemoglobin could not be detected in older lysates with these methods. Its presence results in a dominant type of beta-thalassemia in the two heterozygotes, with moderate anemia, reticulocytosis, nucleated red cells, target cells, and other red cell changes, Heinz body formation, and splenomegaly; the oldest of the two patients was splenectomized. Both subjects had a marked increase in fetal hemoglobin synthesis.

Hb Graz or α2β22(Na2)His→Leu; a new β Chain Variant Observed in four Families from Southern Austria

Two abnormal hemoglobins were accidentally detected by cation exchange high performance liquid chromatography with the Diamat system of Bio-Rad Laboratories; the variants eluted together with the fast-moving Hb A1c. Structural analysis of isolated β chains and sequence analysis of amplified DNA identified a new variant, i.e. Hb Graz that has a His→Leu replacement at position 2 of the β chain, in four healthy, apparently unrelated, adults. The second variant was identical to Hb Sherwood Forest or α2β2104(G6)Arg→Thr; it is believed that this may be the second observation of this abnormal hemoglobin.

Hb CAPA OR α2 94(G1)ASP→GLYβ2, A Mildly Unstable Variant with an A↠G (Gac↠Ggc) Mutation in Cown 94 of the α1-Globin Gene

A 28-year-old female from the city of Kars in Turkey was treated for a chronic iron deficiency anemia with oral iron. During the clinical evaluation a slowly moving hemoglobin (Hb) variant was detected which migrated about like Hb F in alkaline electrophoresis and like Hb S in agar electrophoresis at pH 6.2. The variant appeared mildly unstable in a heat denaturation test (60°C) but the isopropanol stability test was questionably positive (for methodology, see Ref. 1).

Hb Bab-Saaooun or α2β248(CD7)Leu→pro, A Mildly Unstable Variant Found in an Arabian Boy from Tunisia

Hb Bab-Saadoun which has a Leu→Pro substitution at position 48 of the β chain was detected in a young Arabian boy living in Tunisia. His parents did not have the variant which suggests that it occurred as a spontaneous mutation. The substitution is located in the interhelical CD segment; leucine at β48 is an invariable amino acid that may be important as part of a spacer sequence between the two helices and its replacement by proline may affect the stability of the hemoglobin molecule. Hb Bab-Saadoun is unstable in heat and isopropanol stability tests and its chain was best isolated by parachloromercuribenzoate precipitation. It appears unlikely that the presence of Hb Bab-Saadoun results in a hemolytic anemia.

Analysis of Mrna from Red Cells of Patients with Thalassemia and Hemoglobin Variants

During the past decade new procedures have been developed for the isolation of RNA from a few mL of freshly collected blood. This material is reverse transcribed and the resulting cDNA can be used for the determination of the ratios between different types of globin mRNA, namely alpha 2/alpha 1, alpha/zeta, alpha/beta, gamma/beta, beta A/beta X, delta beta Lep/beta, and G gamma/A gamma. Details about these polymerase chain reaction-based methods are reviewed, and information about their usefulness in studying alpha-thalassemia, beta-thalassemia, sickle cell anemia and other beta-globin gene abnormalities, Hb Lepore heterozygosity, and heterozygosity for alpha 2- or alpha 1-globin gene mutations will be provided. The methods are also most useful in characterizing the mRNA types in single, in vitro cultured, BFU-E colonies; in colonies derived from cells of a Hb S heterozygote; for instance, the beta A- and beta(S)-mRNAs were present in all colonies and in about equal quantities, while many of those cells from a subject with a somatic cell mutant (Hb Costa Rica) contained beta A-mRNA and no beta-Costa Rica mRNA, and only a few had both types. The techniques described have considerable diagnostic value and offer a rather simple approach to the study of some of the listed diseases.