Simple PCR detection of haptoglobin gene deletion in anhaptoglobinemic patients with antihaptoglobin antibody that causes anaphylactic transfusion reactions

Abstract

Two anhaptoglobinemic patients showing anaphylactic transfusion reactions by antihaptoglobin antibody were found. Southern blot analysis indicated that 2 patients were homozygous for the deleted allele of the haptoglobin gene (Hpdel) as reported previously. We have identified the junction region of the deletion from genomic DNA of 1 patient using cassette-mediated polymerase chain reaction (PCR). Then, the deleted region from the 5′ breakpoint to the promoter region of the Hpwas amplified from genomic DNA of a control individual using PCR. DNA sequence analysis of these regions indicated that the 5′ breakpoint of the Hpdel allele was located 5.2 kilobase (kb) upstream of exon 1 of the Hp and the 3′ breakpoint was positioned between 52 and 53 base pair (bp) upstream of exon 5 of the haptoglobin-related gene. There was no significant homology between the DNA sequences flanking the 5′ and 3′ breakpoints, except for a 2-bp (TG) identity. To examine the gene frequency, we have developed a simple PCR method to detect the gene deletion. We found 8, 16, and 17 Hpdelalleles in 157 Koreans, 523 Japanese, and in 284 Chinese, respectively, but did not find the Hpdel in 101 Africans or in 100 European-Africans. The incidence of individuals homozygous for the Hpdel allele was therefore expected to be 1/4000 in Japanese, 1/1500 in Koreans, and 1/1000 in Chinese. This incidence is higher than that of IgA deficiency in Japanese. More attention should be paid on haptoglobin deficiency and antihaptoglobin antibody as the cause of transfusion-related anaphylactic reactions in Asian populations.

Current understanding of allergic transfusion reactions: incidence, pathogenesis, laboratory tests, prevention and treatment

“…The allergens that cause allergic transfusion reactions are plasma proteins such as IgA (Vyas et al, 1968;Schmidt et al, 1969;Sandler et al, 1995) and haptoglobin (Hp) (Koda et al, 2000;Shimada et al, 2002). Although there are case reports of anaphylactic reactions, many of which were serious (Vyas et al, 1968;Schmidt et al, 1969;Sandler et al, 1995;Koda et al, 2000;Shimada et al, 2002), there are no reliable estimates regarding the incidence of IgA-and Hp-mediated allergic transfusion reactions. The number of patients who have IgA-or Hp-deficiency and specific antibodies but are never diagnosed because of the absence of symptoms after transfusion remains unknown.…”

The acute phase protein haptoglobin regulates host immunity

“…This is largely a result of the fact that Hp is invariably present and often represents a major constituent of the plasma, lymph, and other extravascular fluids. In humans, only few cases of Hp null patients have been described, which do not present overt immune deficiencies [31,32]. Based on evolutionary considerations, the essential function of Hp in vertebrates is underscored by the strict conservation of its structure and function.…”

A−61C and C−101G Hp gene promoter polymorphisms are, respectively, associated with ahaptoglobinaemia and hypohaptoglobinaemia in Ghana

“…Polymerase chain reaction (PCR) amplification was performed to detect Hp del allele, using the specific primers Hp-del-U and Hp-del-L, as described previously (5). Hp genotypes were determined by PCR, using Hp-Exon-1 U and Hp-Exon-7 L primers (5). Hp 1 variants were also examined by amplifying exons 2-4, using Hp-Exon-2 U and Hp-Exon-4 L primers (5).…”

“…In malaria infection, such benign haemolytic episodes occur accounting partially for reduced Hp levels in individuals within the population (7)(8)(9)(10). The Hp del allele is the only characterized allele for the Hp0 phenotype, but it has not yet been seen in any other population (5). Ahaptoglobinaemia has important clinical consequences.…”