D. D. Pobedimskaya

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.

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 Fannin-Lubbock in Five Spanish Families is Characterized by two Mutations: β111 GTC->CTC (VAL->LEU) AND β119 GGC->GAC (GLY->ASP)

We have sequenced the amplified beta-globin genes of five, apparently unrelated, Spanish adults with a fast-moving hemoglobin variant, and observed a GGC-->GAC mutation at codon 119 which identified the abnormality as Hb Fannin-Lubbock or alpha 2 beta (2)119(GH2)Gly-->Asp. In addition, we found a GTC-->CTC change at codon 111 which leads to a Val-->Leu replacement at this location. Protein analysis of the beta A and beta X chains from one of these individuals confirmed that both mutations are located on the same chromosome. It is hypothesized that some other known variants may carry an additional mutation in one of their exons, resulting in a silent amino acid substitution which may have an effect on some physicochemical property. In the case of Hb Fannin-Lubbock, it appears likely that the Val-->Leu replacement at beta 111, rather than the Gly-->Asp replacement of beta 119, is the cause of the instability of the variant. The Hb Fannin-Lubbock variant in these Spanish families had a normal oxygen affinity.

HB Sinai-Baltimore or α2β218(A15)VAL→GLY, a Silent, Mildly Unstable β Chain Variant Detected by Isoelectrofocusing and High Performance Liquid Chromatography

Isoelectrofocusing and high performance liquid chromatographic methods were used to study an abnormal hemoglobin present in a Black male infant and his mother. The variant, named Hb Sinai-Baltimore, focused slightly behind Hb A and separated incompletely from Hb A by cation exchange high performance liquid chromatography, while the separation of the beta A and beta X chains by reversed phase high performance liquid chromatography was complete. The variant was identified through an analysis of peptides in a tryptic digest of the isolated beta X chain and by sequencing of amplified DNA which included the beta-globin gene. The Val->Gly replacement at position beta 18 (codon 18; GTG->GGG) or at the last position of the A helix decreases the stability of the variant without affecting the hematological parameters of its carrier. The propositus was a compound heterozygote for Hb Sinai-Baltimore and Hb S; the relative quantities of the two variant chains were somewhat different from those of the beta X and beta A chains in the mother with the simple Hb Sinai-Baltimore heterozygosity. An uncertainty about the alpha-globin gene status in the child prevented a further evaluation of these differences.

Hb Ramona or α224(B5)TYR->CYSβ2

Hb Ramona was accidentally detected in a pregnant female of part Spanish descent who was tested during a prenatal physical examination program for the possible presence of a hemoglobin (Hb) variant. Her hematological values were normal: Hb 12.3 g/dl; PCV 0.418 l/l; RBC 4.34 × 1012/l; MCV 96.3 fl; MCH 28.3 pg; MCHC 29.4 g/dl. The variant was detected by isoelectrofocusing (IEF) (1); its mobility was slightly faster than that of Hb A. It could not be separated from Hb A by cation exchange high performance liquid chromatography (HPLC) (2,3), nor did a variant α or β chain separate from the corresponding normal chains by reversed phase HPLC (4,5).

HB Bibba OR α2136(H19)LEU→PROβ2 in a Caucasian Family from Alabama

Several members of a large Caucasian family who presented with a congenital Heinz body hemolytic anemia were found to be carriers of the unstable Hb Bibba or alpha 2 136(H19)Leu-->Pro beta 2. Identification by protein analysis was hampered by the instability of the variant which complicated its isolation from shipped blood samples. Moreover, the detection of the CTG-->CCG mutation at codon 136 of the alpha 2 gene required the substitution of dGTP by dITP during the DNA sequencing process to prevent the occurrence of secondary structures and compressions in the sequencing gel. The first Hb Bibba heterozygote, characterized in 1968 (1), is believed to be a member of this family. The clinical expression of the disease is surprisingly variable.