Elsevier

Clinica Chimica Acta

Volume 433, 10 June 2014, Pages 54-57
Clinica Chimica Acta

Genetic factors associated with serum haptoglobin level in a Japanese population

https://doi.org/10.1016/j.cca.2014.02.029Get rights and content

Highlights

  • The effect of the polymorphisms on serum HP level was examined.

  • rs5472 and HPdel are strong geneic determinants of HP level in Japanese.

  • HPdel heterozygo are useful for determination of genetic factors of HP level.

Abstract

Background

Haptoglobin (HP) is an acute-phase protein induced by inflammatory stimuli. Its serum level varies in several clinical conditions and among individuals. The common HP alleles (HP1 and HP2), HP complete deletion allele (HPdel), and two SNPs (rs5472 and rs2000999) have been reported to be possible genetic determinants of serum HP levels so far. However, no studies have explored the relationship among the polymorphisms using the same samples. For this purpose, the impact of these polymorphisms was examined using Japanese heterozygote samples of the HPdel allele because all of the polymorphisms were found in Japanese samples.

Methods

We collected 194 HPdel heterozygotes and 385 randomly selected samples without HPdel from 5679 Japanese samples. Genotyping of all polymorphisms was performed by PCR using hydrolysis probes. Phenotyping of the common HP alleles was determined by polyacrylamide gel electrophoresis. Serum HP level was measured by a sandwich ELISA.

Results

We observed a significant association between each of the polymorphisms and serum HP level. Two SNPs, rs5472 and rs2000999, were found to be in almost absolute linkage disequilibrium.

Conclusions

We suggest that rs5472 is a strong genetic determinant of HP levels in Japanese samples, in addition to rs2000999, the common HP alleles, and HPdel. Further, the haplotypes of these polymorphisms were determined automatically and the effects of the polymorphisms were clearer in HPdel heterozygotes than samples without HPdel.

Introduction

Haptoglobin (HP) is an acute-phase protein synthesized mainly in the liver; it binds free hemoglobin released by hemolysis or normal red blood cell turnover, thus preventing oxidative damage [1]. The three major subtypes, HP1-1, HP2-1, and HP2-2, are products of two co-dominant alleles, HP1 and HP2, encoded by the HP gene, which is located on chromosome 16. The frequency of the “common alleles” varies among worldwide populations with HP1 frequencies from 0.07 in parts of India to over 0.7 in parts of West Africa and South America [1]. Many diseases have been reported to be associated with the major HP subtypes. Such associations may be due to functional differences between HP1 and HP2 in the binding of hemoglobin and its rate of clearance from serum [1].

The serum level of HP reflects various clinical states such as infectious diseases, malignancy, autoimmune disease, and tissue necrosis. It increases by 200–500% during the seven days after the onset of inflammation [2]. On the other hand, levels decrease during hemolysis, ineffective erythropoiesis, liver disease, and late pregnancy [1], [3]. Recently, measurement of the HP level as one of the inflammatory variables has been shown to improve the prediction of major cardiovascular events [4].

On the other hand, inter-individual variability of the circulating HP level also exists. Gender, age, smoking, and plasma hemoglobin levels were shown to be associated with the HP concentrations in human plasma [5]. In addition, some genetic factors have been reported to affect the HP serum level. Serum HP concentrations are dependent on the major HP subtypes (HP1-1, HP2-1, HP2-2), although they seem to differ among different populations [1]. We previously identified a haptoglobin gene deletion (HPdel) as a causal mutation of anhaptoglobinemia in a Japanese population and inferred the possibility that the allele may be responsible for hypohaptoglobinemia in the heterozygous state, although the sample size was small [6]. The HPdel, which lacks an approximately 28-kb segment that extends from the promoter region to exon 5 of the haptoglobin-related gene, is distributed only in East and Southeast Asian populations with a frequency of 0.009–0.38 [3], [6], [7], [8]. More recently, a genome-wide association study (GWAS) of European children identified one SNP, rs2000999, as a strong genetic determinant of the serum HP level [5], [9]. In a public database (http://www.ncbi.nlm.nih.gov/projects/SNP), this SNP is distributed unequally throughout the global population with a minor allele frequency (MAF) of 0.047–0.476.

Promoter SNPs have also been reported to be associated with HP expression. A SNP at position − 61 (rs5471) was identified as causal polymorphism of HP 2-1 modified phenotypes due to a decreased amount of HP2 polypeptide relative to that of HP1 [10]. The distribution of rs5471 seems to be restricted to Africans and Hispanics (http://www.ncbi.nlm.nih.gov/projects/SNP/) [11]. Recently, HP was identified as one of the genes upregulated in peripheral blood mononuclear cells (PBMCs) of the short-term survival group of advanced castration-resistant prostate cancer (CRPC) patients after cancer peptide vaccination [12]. In addition, a significant correlation between promoter polymorphism at position − 55 (rs5472), and CRPC prognosis or HP expression level was observed in PBMCs before vaccination (personal communication). The MAF of rs5472 seems to be relatively high in many populations (http://www.ncbi.nlm.nih.gov/projects/SNP/) [11]. The gene structures of HP1, HP2, and HPdel and the relative positions of rs5472 and rs2000999 are indicated in Fig. 1.

In this study, we have not only reconfirmed previous findings that common alleles, HPdel and SNP rs2000999 were partly responsible for inter-individual variability of HP, but also found SNP rs5472 located in the promoter region of the HP gene to affect the serum HP level. In addition, we demonstrated the impact of each of the polymorphisms and also observed a direct relationship among the polymorphisms by using the hemizygotes of the HP gene and its flanking sequence.

Section snippets

Materials and methods

This study protocol was approved by the ethics committee of Kurume University School of Medicine.

Association between serum HP level and genetic polymorphisms

We analyzed the association between each of polymorphisms and serum HP level in 194 HPdel heterozygotes and 385 randomly selected samples without HPdel out of 5679 Japanese samples.

First, the samples were divided into two groups according to the presence or absence of HPdel. As shown in Table 1, the median of HP/HPdel was only a quarter of that of HP/HP and the difference between the medians of the two groups was statistically significant. Then we doubled each HP/HPdel value and compared it

Discussion

In this study, all four polymorphisms studied seemed to be associated with serum HP concentration in a Japanese population. However, our samples had no information on potential confounder factors such as age, sex, BMI, biochemical values, or clinical status They are supposed to be considered in conducting association study as in the previous studies [5], [9]. This is the limitation of this study.

A previous analysis of HP2-1 modified phenotypes, which show low HP2 expression, suggested that a

Acknowledgments

We thank Ms. Katherine Ono for the English editing of this manuscript.

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    Current address: Department of Data Science, Kyoto University Hospital, Kyoto 606-8507, Japan.

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