Identification and Diagnosis of Complete Haptoglobin Gene Deletion, One of the Genes Responsible for Adverse Posttransfusion Reactions.

Allergic reactions are the most frequent adverse events in blood transfusion, and anaphylactic shock, although less frequent, is systemic and serious. The cause of allergic reactions to blood transfusions are largely unknown, but deficiencies in serum proteins such as haptoglobin (Hp) can lead to anaphylactic shock. A complete deletion of the haptoglobin gene (HPdel) was first identified in families with anomalous inheritance and then verified as a genetic variant that can cause anaphylactic shock because homozygotes for HPdel have complete Hp deficiency. Thereby, they may produce antibodies against Hp from blood transfusions. HPdel is found in East and Southeast Asian populations, with a frequency of approximately 0.9% to 4%, but not in other populations. Diagnosis of Hp deficiency due to HPdel prior to transfusion is advisable because severe adverse reactions can be prevented by washing the red blood cells and/or platelets with saline or by administering plasma products obtained from an Hp-deficient donor pool. This review outlines the background of the identification of HPdel and several genetic and immunological methods developed for diagnosing Hp deficiency caused by HPdel.

FUT1 variants responsible for Bombay or para-Bombay phenotypes in a database.

Rare individuals with Bombay and para-Bombay phenotypes lack or have weak expression of the ABO(H) antigens on surface of red blood cells due to no or very weak H-type α(1,2)fucosyltransferase activity encoded by FUT1. These phenotypes are clinically important because subjects with these phenotypes can only accept transfusions of autologous blood or blood from subjects with the same phenotypes due to the anti-H antibody. To survey FUT1 alleles involved in Bombay and para-Bombay phenotypes, the effect of 22 uncharacterized nonsynonymous SNPs in the Erythrogene database on the α(1,2)fucosyltransferase activity were examined by transient expression studies and in silico analysis using four different online software tools. Two nonfunctional alleles (FUT1 with c.503C>G and c.749G>C) and one weakly functional allele (with c.799T>C) were identified in transient expression studies, while the software predicted that the proteins encoded by more alleles including these would be impaired. Because both nonfunctional FUT1 alleles appear to link to the nonsecretor alleles, homozygotes of these alleles would be of the Bombay phenotype. The present results suggest that functional assays are useful for characterization of nonsynonymous SNPs of FUT1 when their phenotypes are not available.

Detection of c.375A>G, c.385A>T, c.571C>T, and sedel2 of FUT2 via Real-Time PCR in a Single Tube.

α(1,2)fucosyltransferase (Se enzyme) encoded by FUT2 is involved in the secretor status of ABH(O) blood group antigens. The sedel2 allele is one of the non-functional FUT2 (se) alleles in which 9.3 kb, containing the entire coding region of FUT2, is deleted by Alu-mediated nonhomologous recombination. In addition to this allele, three SNPs of FUT2, c.375A>G, c.385A>T, and c.571C>T, appear to be prevalent in certain Oceanian populations such as Polynesians. Recently, we developed an endpoint genotyping assay to determine sedel2 zygosity, using a FAM-labeled probe for detection of the sedel2 allele and a VIC-labeled probe for the detection of FUT2. In this study, instead of the VIC probe, a HEX-labeled probe covering both c.375A>G and c.385A>T and a Cy5-labeled probe covering c.571C>T were added to the sedel2 allele assay mixture to allow for the simultaneous detection of these four variations via endpoint genotyping for sedel2 zygosity and fluorescence melting curve analysis for c.375A>G, c.385A>T, and c.571C>T genotyping. The results obtained from 24 Samoan subjects using this method were identical to those obtained using previous methods. Therefore, it appears that the present method can accurately determine these four variations simultaneously.

Estimation of Lewis Blood Group Status by Fluorescence Melting Curve Analysis in Simultaneous Genotyping of c.385A>T and Fusion Gene in FUT2 and c.59T>G and c.314C>T in FUT3.

Lewis blood group status is determined by two fucosyltransferase activities: those of FUT2-encoded fucosyltransferase (Se enzyme) and FUT3-encoded fucosyltransferase (Le enzyme). In Japanese populations, c.385A>T in FUT2 and a fusion gene between FUT2 and its pseudogene SEC1P are the cause of most Se enzyme-deficient alleles (Sew and sefus), and c.59T>G and c.314C>T in FUT3 are tag SNPs for almost all nonfunctional FUT3 alleles (le59, le59,508, le59,1067, and le202,314). In this study, we first conducted a single-probe fluorescence melting curve analysis (FMCA) to determine c.385A>T and sefus using a pair of primers that collectively amplify FUT2, sefus, and SEC1P. Then, to estimate Lewis blood group status, a triplex FMCA was performed with a c.385A>T and sefus assay system by adding primers and probes to detect c.59T>G and c.314C>T in FUT3. We also validated these methods by analyzing the genotypes of 96 selected Japanese people whose FUT2 and FUT3 genotypes were already determined. The single-probe FMCA was able to identify six genotype combinations: 385A/A, 385T/T, sefus/sefus, 385A/T, 385A/sefus, and 385T/sefus. In addition, the triplex FMCA successfully identified both FUT2 and FUT3 genotypes, although the resolutions of the analysis of c.385A>T and sefus were somewhat reduced compared to that of the analysis of FUT2 alone. The estimation of the secretor status and Lewis blood group status using the form of FMCA used in this study may be useful for large-scale association studies in Japanese populations.

Fluorescence Melting Curve Analysis for Concurrent Genotyping of Three Tag SNPs in FUT3.

The synthesis of Lewis blood group antigens is governed by two fucosyltransferase genes, FUT2 and FUT3. Evidence is accumulating to suggest that functional polymorphisms of FUT2 and FUT3 are associated with a variety of clinical conditions. Fluorescence melting curve analysis (FMCA), using three different dual-labeled probes for concurrent genotyping of three single nucleotide polymorphisms (SNPs) of FUT3, c.59T>G, c.314C>T, and c.484G>A for Lewis-negative allele inference, was developed and validated using Ghanaian and Caucasian subjects. Although two other SNPs, c.55G>A, and c.61C>T, are located in the probe sequence for c.59T>G, it seems feasible to detect these two SNPs along with c.59T>G. The results obtained by probe-based FMCA were in perfect accordance with those obtained by Sanger sequencing for 106 Ghanaians and 100 Caucasians. The present method is useful and reliable for estimating Lewis-negative alleles on a relatively large scale.

Duplex dual-labeled fluorescence probe-based melting curve and endpoint genotyping assays for genotyping of rs2000999 and haptoglobin gene deletion.

Haptoglobin (Hp) is a hemoglobin-binding serum glycoprotein. Some variations in the Hp gene (HP) or Hp-related gene (HPR), including a single-nucleotide polymorphism in intron 2 of HRP, rs2000999, and a complete deletion of the HP gene (HPde l ), one of the rare variants of HP, have been reported to correlate with the serum cholesterol concentration as well as the serum Hp concentration. In this study, we developed a duplex dual-labeled fluorescence probe-based method to simultaneously determine the rs2000999 G > A polymorphism by melting curve genotyping and the zygosity of HPde l by endpoint genotyping. This method was then validated by using the genomic DNA from 94 Japanese subjects for whom genotypes of rs2000999 and HPdel zygosity had already been determined. The results obtained with this method were in perfect agreement with the previous ones. Thus, the present method enables us to estimate these two polymorphisms in relatively large-scale groups of subjects, especially in Asian populations where the HPdel is distributed.

Simultaneous genotyping of three major Se enzyme inactivating SNPs of FUT2 based on a triplex probe-based fluorescence melting-curve analysis.

BACKGROUND: The ABO(H) secretor status is controlled by FUT2-encoded α(1,2)fucosyltransferase (Se enzyme) activity. Three SNPs of FUT2, 302C>T (rs200157007), 385A>T (rs1047781), and 428G>A (rs601338), cause three major variants of nonsecretor (se) or weak-secretor (Sew) alleles. Evidence has been accumulating that suggests the secretor status is associated with various conditions including infectious diseases but a robust multiplex method for assaying relatively large-scale samples to determine the genotype of these three SNPs simultaneously has not been developed yet. METHODS: By combined usage of two Eprobes and a dual-labeled fluorescence probe, we developed a real-time PCR, followed by triplex probe-based fluorescent melting-curve analysis (FMCA) for genotyping of 302C>T, 385A>T, and 428G>A of FUT2 in a single tube. RESULTS: Three genotypes of each of three variants of FUT2 were accurately determined by the triplex probe-based FMCA. We then validated this method using genomic DNA samples of 47 Bangladeshis, and the results obtained by using this method were fully concordant with those by previous Sanger sequencing. CONCLUSIONS: Since the present single triplex probe-based FMCA is robust, fast, and cost-effective, we are able to effectively estimate the secretor status of subjects on a large scale in many populations around the world.

Detection of five common variants of ABO gene by a triplex probe-based fluorescence-melting-curve-analysis.

Current studies have suggested that the ABO blood group system is associated with several clinical conditions. For large-scale genotyping of ABO alleles, we developed a triplex fluorescence melting curve analysis (FMCA) to determine five single nucleotide variants (SNVs), c.261delG, c.796C>A, c.802G>A and c.803G>C and c.1061delC, responsible for common ABO phenotypes using dual-labeled self-quenched (TaqMan) probes in a single tube. We accurately determined c.796C>A, c.802G>A, and c.803G>C genotypes using a FAM-labeled probe, c.261delG using a CAL Fluor Orange 560- labeled probe, and c.1061delC using a Cy5-labeled probe. The present genotyping results of five SNVs in 214 subjects of the 1000 Genomes Project were in full agreement with those of the database sequence. The predicted ABO phenotypes using combinations of these five SNVs by this method in 288 Japanese subjects were in complete agreement with those by hemagglutination assay, although we did not find any A2 (alleles containing c.1061delC) or O.02 (alleles containing c.802G>A) alleles. The present triplex probe-based FMCA is a valid and credible method for a considerably accurate large-scale determination of ABO allele genotypes and estimation of phenotypes.

Rapid genotyping of 508G>A (rs3745635) and 1067T>A (rs3894326) of FUT3 by a duplex Eprobe-mediated melting curve analysis.

BACKGROUND AND OBJECTIVES: Lewis histo-blood group phenotypes are regulated by the action of FUT3-encoded α(1,3/1,4)fucosyltransferase and FUT2-encoded α(1,2)fucosyltransferase. Since Lewis phenotypes are suggested to be associated with various clinical conditions, a method for large-scale FUT3 genotyping is desirable. In worldwide populations, 508G>A and 1067T>A of FUT3 are two of three common causal single nucleotide polymorphisms for Lewis-negative alleles. MATERIALS AND METHODS: We developed a duplex Eprobe-mediated melting curve analysis for genotyping 508G>A and 1067T>A simultaneously and applied this method to 106 Ghanaian and 140 Japanese subjects. RESULTS: The results of both 508G>A and 1067T>A genotyping by duplex Eprobe-mediated melting curve analysis were completely in agreement with the results of a DNA sequence analysis in 106 Ghanaians and polymerase chain reaction-restriction fragment length polymorphism analysis in 140 Japanese subjects. CONCLUSION: The present duplex Eprobe-mediated melting curve analysis is valid and credible for large-scale estimation of Lewis-negative alleles.

Estimation of Lewis-negative alleles by high-resolution melting analysis of three tag SNPs of FUT3.

BACKGROUND AND OBJECTIVES: The expression of type 1 chain Lewis blood group antigens is regulated by secretor-type α(1,2)fucosyltransferase, encoded by FUT2, and Lewis α(1,3/1,4)fucosyltransferase, encoded by FUT3. Accumulating evidence has linked Lewis phenotypes or genotypes to various clinical conditions. Thus, in addition to FUT2, large-scale FUT3 genotyping is important. Because FUT3 has two paralogous genes (FUT5 and FUT6) with high DNA sequence similarity, we should select the polymerase chain reaction (PCR) primers carefully for FUT3 genotyping. Previously, we suggested that 13G>A (rs28362458), 59T>G (rs28362459) and 202T>C (rs812936) could be selected as tag single nucleotide polymorphisms (SNPs) for detection of Lewis-negative alleles (le). MATERIALS AND METHODS: In this study, three high-resolution melting (HRM) analyses for genotyping these SNPs were developed and applied for 140 Japanese, eight Ghanaians and four Sinhalese subjects. RESULTS: Each of three genotypes of 13G>A (G/G, G/A, A/A), 59T>G (T/T, T/G, G/G) and 202T>C (T/T, T/C, C/C) was discriminated clearly. Although we need to be careful in interpretation of results due to SNPs other than the 59T>G in the amplicon, the results of 59T>G genotyping were in full agreement with the results by a previous PCR-restriction fragment length polymorphism analysis in 140 Japanese. In addition, three heterozygotes of 202C substitution were identified, and no one having a 13A substitution was found in 140 Japanese. CONCLUSION: The present HRM analyses are useful and reliable methods for large-scale estimation of le alleles.

Real-time PCR-based detection of the Alu-mediated deletion of FUT2 (sedel2).

Secretor status of the ABH(O) histoblood group antigens is regulated by secretor type α(1,2)fucosyltransferase encoded by FUT2. The sedel2 allele is a complete deletion of the FUT2 coding region generated by Alu-mediated homologous recombination. This deletion seems to be exclusively encountered in certain Oceanian populations. From the perspective of forensic science, sedel2 is considered to be one of ancestry informative markers for these populations. Real-time PCR followed by melting curve analysis was employed to find primer set to specifically amplify sedel2. We designed primers which produced a 231-bp amplicon specific to sedel2. The specificity of these primers was also confirmed by gel electrophoresis and sequencing of the PCR product. Then, two real-time PCR methods based on melting curve analysis and a hydrolysis probe were designed to determine sedel2 zygosity by adding FUT2-specific primers. These two methods were validated by analyzing 24 Samoan subjects. The results obtained from 24 Samoan subjects by the two methods were fully in accordance with those obtained by a previous conventional PCR method that amplified a 2.7-kb fragment of sedel2. Therefore, these two methods seemed to accurately determine the zygosity of sedel2 and were useful for investigation of the distribution and origin of this deletion.

Rapid detection of phenotypes Bombay sedel and nonsecretor rs200157007 SNP (302C > T) by real-time PCR-based methods.

The sedel allele is one of the nonsecretor alleles (se) of FUT2 generated by an Alu-mediated recombination event and was first found in Indian Bombay phenotype individuals who have anti-H, anti-A, and anti-B antibodies in their serum. As well as anti-A, and anti-B antibodies, anti-H is clinically significant because it causes sever hemolytic transfusion reactions. Like sedel, se302 having a missense single nucleotide polymorphism (SNP), 302C > T, is characteristic of South Asians with a frequency of 10-30%. We developed a real-time PCR melting curve analysis for detection of sedel using a 127-bp amplicon encompassing the breakpoint junction. In addition, by performing duplex PCR by amplifying a 65-bp amplicon of the FUT2 coding region at the same time, we could determine the zygosity of sedel in a single tube. We also developed an Eprobe-mediated PCR assay (Eprobe-PCR) for detection of 302C > T of FUT2. These methods were validated by analyzing 58 Tamils and 54 Sinhalese in Sri Lanka. Both the duplex PCR melting curve analysis for determination of sedel zygosity and the Eprobe-PCR assay for detection of 302C > T exactly determined three genotypes. In addition, the results of the present methods were in complete agreement with those obtained by previously established methods. The two present methods were reliable and seem to be advantageous for large-scale association studies of FUT2 polymorphisms in South Asian populations.

Detection of the weak-secretor rs1047781 (385A>T) single nucleotide polymorphism using an unlabeled probe high-resolution melting-based method.

FUT2 encodes galactoside 2-α-l-fucosyltransferase 2 which determines the secretor status of ABO(H) blood group antigens. Secretors have at least one functional FUT2 allele (Se), while nonsecretors or weak secretors are homozygous for nonfunctional (non- or weak secretor) FUT2 alleles (se or Sew ). The Sew having the 385A>T missense SNP (rs1047781) is the prevalent nonfunctional allele in East and Southeast Asians. In this study, we developed an unlabeled-probe high-resolution melting (HRM) analysis for genotyping of 385A>T and validated the method by analyzing 72 Japanese whose 385A>T genotypes were confirmed by DNA sequencing. The unlabeled-probe HRM analysis clearly discriminated three genotypes of 385A>T. In addition, the results obtained for the 72 Japanese by this method were fully concordant with previous ones. Estimation of secretor status using this cost-effective method may be useful in East and Southeast Asian populations.

Estimation of secretor status of ABO antigens by high-resolution melting analysis of rs601338 (428G > A).

BACKGROUND: FUT2 determines the secretor status of ABH antigens. Many lines of evidence suggest an association between secretor status and susceptibility to various clinical conditions. For this kind of study, large-scale genotyping of FUT2 is necessary. Because FUT2 has a pseudogene (SEC1) with high DNA sequence similarity and is rich in population-specific SNPs, we need to pay attention in designing the primers for genotyping FUT2. The se428 allele having a 428G > A nonsense SNP (W143X, rs601338) is the predominant non-secretor allele in Europeans, Latin Americans and Africans. On the other hand, se357,480,778del having the 778C > del frameshift SNP (P260Lfs*16, rs1799761) is almost exclusively found in Africans with frequencies of 1-4%. STUDY DESIGN AND METHODS: We developed high-resolution melting (HRM) analyses using short (69-bp for 428G > A, 65-bp for 778C > del) amplicons for genotyping two SNPs directly and validated the method by analyzing 95 Ghanaians whose FUT2 genotypes were previously determined. RESULTS: Two sets of assays clearly discriminated three genotypes of 428G > A (G/G, G/A, A/A), and two genotypes of 778C > del (C/C, C/del). In addition, the results obtained for the 95 Ghanaians by HRM analysis were in full agreement with previous ones. CONCLUSION: The present HRM analysis reliably genotyped 428G > A. Thus, estimation of secretor status based on se428 using the present HRM analysis may be useful for large scale association studies of FUT2. In addition to 428G > A, genotyping of other causal polymorphisms for non-secretors with high frequency, as is the case with 778C > del for Africans, is desirable for more accurate estimation of the secretor status of the target populations.

Survey and characterization of nonfunctional alleles of FUT2 in a database.

The expression of ABO antigens in human saliva is regulated by the FUT2 gene, which encodes a secretor type α(1,2)fucosyltransferase. Secretors express ABO substrates in saliva and non-secretors do not. Secretor status is an object of concern, especially for susceptibility to various infectious diseases. A multitude of single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) have been reported, and they show unique distributions among different populations. In this study, we selected 18 uncharacterized FUT2 alleles listed in the Erythrogene database and obtained genomic DNA having these alleles. We experimentally confirmed the haplotypes, but 10 of 18 alleles disagreed with those in the database, which may be attributed to their low frequency. We then examined the activity of the encoded α(1,2)fucosyltransferase for 13 alleles by flow cytometry of H antigen expression. The impact of each nonsynonymous SNP on the enzyme was also estimated by software. We finally identified two non-secretor alleles (se610and se357,856,863) and one weak secretor allele (se262,357), while in silico analysis predicted that many alleles impair the function. The present results suggest that correct haplotyping and functional assays are desirable for analysis of the FUT2 gene.

Detection of helium in a fire victim: A case report.

We report here detection of helium in specimens derived from a burn autopsy case. A male was found in a burnt bedroom. Part of a heat-denatured plastic bag, sealing tape, and flexible tubing remained on his head and neck. In addition, five helium tanks were found near him. His history in conjunction with the discovery conditions suggested a suicide attempt by inhalation of helium. The body had extensive first to fourth degree burns caused by heat. A small amount of soot was deposited in the respiratory tract. Except for the thermal burns, no other injuries were found. Toxicologically, the blood carboxyhemoglobin saturation levels were less than 6%, while combustion-derived volatile hydrocarbons such as benzene or toluene were detected in the blood. In addition, tracheal gas, gastric gas, headspace gas of lung tissue, brain, and heart blood were collected during autopsy for detection of helium. Analysis was performed using headspace gas chromatography with a thermal conductivity detector. Helium was detected in all of the samples tested. Etizolam at a low limit of therapeutic concentration or less was detected in the blood. Neither ethanol nor other drugs of abuse were detected in his blood or urine. Autopsy findings and experiments suggest that the victim inhaled helium and was still alive when a fire broke out. The cause of his death was diagnosed as death from fire and flames. The present result suggests that helium may remain in a burned body and that investigation of helium in cases of fire-related deaths is informative for determination of the cause of death or confirmation of the ante mortem involvement of helium.

Effect of tolvaptan on renal involvement in patients with autosomal dominant polycystic kidney disease according to different gene mutations.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is an inherited disorder caused by mutations in the polycystic kidney disease (PKD) gene. Although tolvaptan has benefits for renal involvement, the different effects depending on the gene mutation type are unknown. Thus, we explore the different effects of tolvaptan on the annual changes in total kidney volume (%TKV) and estimated glomerular filtration rate (eGFR) according to the gene mutation type in ADPKD patients. METHODS: In total, 135 ADPKD patients were screened, and 22 patients taking tolvaptan for at least a year were retrospectively studied at the Kurume University Hospital. We examined the decline in renal function and %TKV by computed tomography and analyzed the gene mutation. Patients were classified into the following four groups according to gene mutation type: PKD1-truncated, PKD1-non-truncated, PKD2, and mutation not found. Patients were treated with tolvaptan, and the effects of tolvaptan were analyzed according to the gene mutation type. RESULTS: Patients (age: 52.3 ± 11.2 years) were administered tolvaptan at a dose of 45 or 60 mg. No variation was observed in the annual changes in eGFR (%eGFR) (before: - 10.5% ± 13.9%, after: - 14.4% ± 8.1%, P = 0.139), whereas %TKV was significantly improved after the tolvaptan treatment (before: 14.9% ± 8.0%, after: - 5.4% ± 7.6%, P < 0.001). Unlike %eGFR, tolvaptan treatment significantly improved %TKV, regardless of the type of gene mutation. CONCLUSIONS: A year treatment with tolvaptan significantly improved %TKV in patients with ADPKD, regardless of the gene mutation type.

High-resolution melting analysis for detection of fusion allele of FUT2.

The secretor status of ABH antigens, determined by FUT2 polymorphisms, affects susceptibility to various infectious diseases. In addition to many SNPs responsible for the nonsecretor phenotype, five nonfunctional alleles (se) resulting from copy number variations have been reported. One of the five alleles generated by an unequal crossover between FUT2 and a pseudogene (SEC1), is sefus . This allele may be misidentified as a functional allele if only common inactivating SNPs are genotyped because it contains the 3' region of the functional FUT2. Therefore, accurate detection of sefus is desirable. For this purpose, a high-resolution melting (HRM) analysis is developed for detection of sefus in which a 284bp fragment of SEC1 and sefus but not FUT2, are amplified. This HRM analysis detected sefus reliably. Thus, an initial screening or prescreening for sefus using HRM analysis seems to be useful for association studies of FUT2.

Haptoglobin polymorphisms in Latin American populations.

Several genetic polymorphisms of the haptoglobin gene (HP) or haptoglobin-related gene (HPR) were reported to show a population-specific distribution and to be associated with not only serum haptoglobin (HP) but also cholesterol levels. For such association studies, it is important to know the distribution of polymorphisms or their haplotypes in the populations concerned. However, no comprehensive genetic studies have explored this in Latin Americans, and not every human variation or genotype is available in a database. In this study, we determined the genotypes of common HP (HP1 and HP2), HPdel, rs5471, rs5472, and rs2000999 in several Latin American populations. Haplotypes of rs5472-common HP-rs2000999 polymorphisms were estimated. We did not encounter any HPdel, and the frequencies of rs5471 A, rs5472 A, HP1, and rs2000999 G were higher than their counterpart alleles in studied populations. All of the alleles with higher frequency in the Latin Americans are associated with higher serum HP and lower cholesterol levels. Both A-1-G (probably HP1S) and G-1-G (probably HP1F) haplotypes were higher in Latin American populations than those in other geographic regions. In addition, the genetic influx from populations of other continents into Peruvians seems to be relatively lower than that into other Latin Americans.

Serum haptoglobin correlates positively with cholesterol and triglyceride concentrations in an obese Mongolian population.

BACKGROUND: Recent studies revealed that several genetic polymorphisms of haptoglobin gene (HP) and the haptoglobin-related protein gene (HPR) associated not only with haptoglobin (HP) but total, non-HDL, and/or LDL cholesterol concentrations in various populations. METHODS: Association between serum HP concentrations and polymorphisms of HP and the HPR gene, or anthropometric and metabolic factors were examined in Mongolian participants (n = 927) using linear regression analyses. RESULTS: The association of HP and HPR polymorphisms with serum HP concentration but not serum lipids concentrations was observed. However, subgroup analysis revealed that the association of HP and HPR polymorphisms with serum HP concentration was weakened in subgroup of obese (BMI ≥ 30) subjects and positive correlations between serum HP and non-HDL cholesterol, HDL cholesterol or triglyceride concentrations were observed in the obese subjects as compared with in subgroups of normal weight (BMI < 25) and overweight (25 ≤ BMI < 30) subjects. CONCLUSION: The degree of obesity strongly affects the relationships between serum HP concentrations and several genetic, anthropometric and metabolic factors. These results suggested that we need to take into account the degree of obesity when considering the HP polymorphisms as predictive markers for clinical states.