Haemoglobin Lleida: a new α₂‐globin variant (12 bp deletion) with mild thalassaemic phenotype

Abstract

Molecular studies of alpha-thalassaemias have revealed defects at different steps in the process of alpha-gene expression. It is not surprising, therefore, that in some cases a single mutation or small deletion can result in a structurally abnormal haemoglobin that produces the alpha-thalassaemia phenotype. In this report we describe a new unstable alpha-globin variant, Hb Lleida, in a Spanish patient with alpha-thalassaemia trait. The mutation was detected by single-strand conformation polymorphism in the third exon of the alpha 2-globin gene. Direct sequence analysis of the alpha-globin gene showed a 12 bp deletion as the only defect of the alpha 2- and alpha 1-globin genes. The propositus was revealed to be a heterozygous carrier, and two alleles were separated by electrophoresis. This deletion causes the loss of four aminoacid residues (from codon 113 to 116) and would be expected to produce an unstable haemoglobin, as a shorter alpha-globin chain variant is created with 137 amino acids instead of 141 amino acids present in a normal alpha-globin chain. However, no abnormal haemoglobin was found by either isoelectric focusing or haemoglobin electrophoresis. Since the deletion affects an aminoacid residue (114 Pro) involved in alpha 1-beta 1-globin chain contacts, the interaction required for efficient Hb assembly is also compromised. The resulting unstable alpha-globin chain is rapidly catabolized and unsuitable for haemoglobin tetramer formation, causing an alpha-thalassaemia trait phenotype in the heterozygous patient.

α‐thalassaemia due to a single codon deletion in the α‐1‐globin gene. Computational structural analysis of the new α‐chain variant

“…Since none of the known -thalassaemia determinants was found, the complete 1 and 2 -globin genes were amplified by polymerase chain reaction (PCR) using previously described primers (Dodé et al, 1990). An aliquot of these PCR products was used as a template to amplify the exon and intron boundaries into three different fragments for each gene, using a combination of specific primers (Ayala et al, 1996b). Then, these fragments were submitted to non-radioactive SSCP analysis as described previously (Ayala et al, 1996b).…”

“…An aliquot of these PCR products was used as a template to amplify the exon and intron boundaries into three different fragments for each gene, using a combination of specific primers (Ayala et al, 1996b). Then, these fragments were submitted to non-radioactive SSCP analysis as described previously (Ayala et al, 1996b). The region of the -globin gene showing an abnormal mobility by SSCP was reamplified by using specific flanking primers, one of which was biotinylated at the 5' end.…”

FIRST DESCRIPTION OF A FRAMESHIFT MUTATION IN THE α1‐GLOBIN GENE ASSOCIATED WITH α‐THALASSAEMIA

“…When the above screen was negative, the entire a 1 -and a 2 -globin genes were submitted to non-radioactive PCR-SSCP analysis as described previously (Ayala et al, 1996b), for rapid detection of point mutations, short deletions or insertions. Fragments showing an abnormal SSCP band pattern with a mobility shift or the presence of extra bands, indicate the possible existence of some gene abnormality.…”

“…This new mutation reduces the aglobin output and leads to the mild a-thalassaemic phenotype in the affected subject. In addition to the intrinsic interest of this case, our study demonstrated that the nonradioactive single-strand conformation polymorphism (SSCP) analysis, devised in our laboratory during the last two years (Ayala et al, 1996b), is a powerful tool for the routine detection of specific mutations in a-thal.…”

Screening for mutations in human alpha-globin genes by nonradioactive single-strand conformation polymorphism

“…α-genes: Ayala et al (5), used the singlestrand conformation polymorphism (SSCP) technique described by Orita et al (6), and Harteveld et al (7) used SSCP in combination with denaturating gradient gel electrophoresis (DGGE) (8). These two methods, however, present some disadvantages because the former is a radioactive SSCP and the latter combines two techniques and two different types of equipment, with the DGGE Figure 1.…”