Caucasian Blood Donors Research Articles

CCR5 promoter polymorphism determines macrophage CCR5 density and magnitude of HIV-1 propagation in vitro

The common CCR5 promoter polymorphism at position −2459 (A/G) has been associated with differences in the rate of progression to AIDS, where HIV-1-infected individuals with the CCR5 −2459 G/G genotype exhibit slower disease progression than those with the A/A genotype. Mechanisms underlying the relationship between these polymorphisms and disease progression are not known. Here through in vitro infection of peripheral blood mononuclear cells obtained from healthy Caucasian blood donors with macrophage-tropic HIV-1 isolates we observed low, medium, and high viral propagation in association with G/G, A/G, and A/A promoter genotypes, respectively. Flow cytometric analysis of unstimulated CD14+ monocytes from these same donors revealed a similar hierarchy of CCR5 receptor density in association with promoter genotypes. Finally, PBMC from persons with the G/G promoter polymorphism produced higher levels of β-chemokines after in vitro stimulation. Thus, the CCR5 −2459 (A/G) promoter polymorphism determines CCR5 expression and predicts the magnitude of HIV-1 propagation in vitro. These findings may provide important insight regarding the regulation of mechanisms that influence the rate of HIV-1 propagation and progression to AIDS.

Prevalence of factor VII deficiency and molecular characterization of the F7 gene in Brazilian patients.

The prevalence of factor VII (FVII) deficiency in 267 Brazilian patients was estimated to be 4.1%, including one patient with significant bleeding, five with minor bleeding and five patients asymptomatic. Only one novel mutation 8926G <-- T (I140S) was seen in one patient. The other mutations were 10828G <-- A (R304Q) in three patients, 10846G <-- T (C310F) in one patient, and 10909G <-- A (G331D) in one patient. Except for one homozygous patient (C310F) with a severe deficiency, only one allele was affected in all other instances. An inverse association between F7 polymorphisms and FVII activity were found in these patients, as those with higher levels of FVII activity presented the genotype described in the literature as related to reduced FVII activity. As the R304Q mutation was the most frequent in these patients, and may be associated with an asymptomatic form of the disease, particularly in Blacks, we examined this mutation and FVII activity in 49 Blacks and 49 Caucasian blood donors with no clinical bleeding. None of the individuals showed the R304Q mutation, and FVII activity was normal in all of them, thus indicating that FVII deficiency is not common in normal individuals of these two ethnic groups in Brazil. This is the first study in South America to examine the prevalence and molecular basis of FVII deficiency, including the description of a novel mutation.

CYP3A5 Variant Allele Frequencies in Dutch Caucasians

Enzymes of the cytochrome P450 3A (CYP3A) family are responsible for the metabolism of >50% of currently prescribed drugs. CYP3A5 is expressed in a limited number of individuals. The absence of CYP3A5 expression in approximately 70% of Caucasians was recently correlated to a genetic polymorphism (CYP3A5*3). Because CYP3A5 may represent up to 50% of total CYP3A protein in individuals polymorphically expressing CYP3A5, it may have a major role in variation of CYP3A-mediated drug metabolism. Using sequencing, have been identified (Hustert et al. Pharmacogenetics 2001;11:773-9; Kuehl et al. Nat Genet 2001;27:383-91) variant alleles *2 through *7 for CYP3A5. Detection of CYP3A5 variant alleles, and knowledge about their allelic frequency in specific ethnic groups, is important to establish the clinical relevance of screening for these polymorphisms to optimize pharmacotherapy. In a group of 500 healthy Dutch Caucasian blood donors, we determined the allelic frequency of the CYP3A5*2, *3, *4, *5, *6, and *7 alleles by use of newly developed PCR-restriction fragment length polymorphism assays. The frequency of the defective CYP3A5*3 allele in the Dutch Caucasian population was 91%, followed by the CYP3A5*2 (1%) and CYP3A5*6 (0.1%) alleles. The CYP3A5*4, *5, and *7 alleles were not detected. On the basis of its allelic frequency, screening for the CYP3A5*3 allele in the Caucasian population is extremely relevant. In addition, screening for the CYP3A5*2 allele may be taken into consideration in individuals heterozygous for the CYP3A5*3 allele. The CYP3A5*4, *5, *6, and *7 alleles have low allelic frequencies that do not support initial screening.

Determination of the genomic structure and mutation screening in schizophrenic individuals for five subunits of the N-methyl-D-aspartate glutamate receptor.

The glutamatergic system is the major excitatory neurotransmitter system in the CNS. Glutamate receptors, and in particular N-methyl-D-aspartate (NMDA) receptors, have been proposed as mediators of many common neuropsychiatric phenotypes including cognition, psychosis, and degeneration. We have reconstructed the genomic structure of all five genes encoding NMDA receptors in silico. We screened each for sequence variation and estimated the allele frequencies of all detected SNPs in pooled samples of 184 UK Caucasian schizophrenics and 184 UK Caucasian blood donor controls. Only a single non-synonymous polymorphism was found indicating extreme selection pressure. The rarity of non-synonymous changes suggests that such variants are unlikely to make a common contribution to common phenotypes. We found a further 26 polymorphisms within exonic or adjacent intronic sequences. The minor alleles of most of these have a relatively high frequency (63% above 0.2). These SNPs will therefore be suitable for studying neuropsychiatric phenotypes that are putatively related to NMDA dysfunction. Pooled analysis provided no support for association between any of the GRIN genes and schizophrenia.

CD14 and TNFα promoter polymorphisms in patients with acute arthritis

Objective. To examine CD14 and TNF f gene polymorphisms in early arthritis in relation to clinical outcome. Methods. We studied 141 Caucasians who had had early arthritis 10 to 38 years earlier. We analysed CD14 (-159) and TNF f (- 238, - 308, - 376) polymorphisms using a novel cycle minisequencing method. DNA pools from 370 Caucasian blood donors served as controls. Results. CD14 (-159)C M T allele frequencies were comparable among patients and controls (39% vs 40%). Fifty men and 42 women had recovered while 24 men and six women had chronic spondyloarthropathy (SpA). Mutant T allele frequency was higher in the chronic SpA group than in the recovered group in women (75% vs 32%, relative risk 1.3, 95% confidence limit 1.1 to 1.6, P= 0.011), but not in men (38% vs 44%). All female patients with chronic SpA had CD14 (- 159)T allele and none had a possibly protective TNF f (- 308)G M A allele. Conclusion. Possession of CD14 (- 159)T allele does not increase risk of ReA but may increase susceptibility of female patients to development of chronic SpA.

Rapid genotyping of the major alleles at the Duffy (FY) blood group locus using real-time fluorescence polymerase chain reaction

Abstract The Duffy blood group system has clinical importance due to involvement in transfusion reactions and hemolytic disease of the newborn. Recently, the molecular basis of the two alleles, FY*A and FY*B (125G&gt;A), and the mutation situated in the promoter region of the FY gene (–33T&gt;C), have been elucidated. In order to develop an accurate, easy, and rapid genotyping method, we describe a procedure using the LightCycler®. Samples from 53 Caucasian Portuguese blood donors and 7 black, healthy, European individuals were phenotyped with commercial antisera. DNA was extracted from blood samples and the relevant sequences were amplified with the same cycling conditions, using real-time polymerase chain reaction. The melting point of the FY*A allele was 63°C and of the FY*B allele, 55°C. The allele without mutation at the promoter region had a melting point at 64°C and the FY*B silent allele at 58°C. The results in Caucasian individuals were similar to those found in European and American populations. When FY genotyping techniques are necessary, the methodology described is preferable to conventional methods as it is reliable, high speed, and uses small volumes, providing a highly competitive technology for use by a routine laboratory. Immunohematology 2001;17:42–44.

Transfusion des patients atteints d'hémoglobinopathies

A-Thalassemia involves a production deficiency concerning the synthesis of alpha-globin chains, and beta-thalassemia involves the beta-globin chains. Only a few patients in France are affected by the major form of thalassemia (certain types of homozygotic beta-thalassemia). Also, the systematic screening of 'at-risk' couples and prenatal diagnosis has helped to considerably reduce the incidence of new cases. The decision to perform regular blood transfusions is made when Hb levels fall below values that are compatible with normal activity. Hb levels above 10 g/dL permit normal educational, recreational and professional activity. This level is generally maintained via a 15 mL/kg erythrocyte concentrate supplement every three weeks, or 20 mL/kg every four weeks. However, the appearance of antierythrocytic autoantibodies is possible, and this may also result in an increase in blood transfusion requirements. In intermediate thalassemia patients, residual Hb levels are maintained at between 7 and 10 g/dL, and transfusion of erythrocyte concentrates is only made in the case of aggravation of chronic anemia or when there are signs of intolerance to chronic anemia. In France, there is relatively large population of patients with sickle cell disease. Blood transfusion is a major element in the treatment of these patients. Simple transfusion is performed in cases of a lack of iron or folates, increased hemolysis, splenic sequestration or parvovirus 19 infection. The target hematocrit should mostly remain at the patient's baseline value. Exchange transfusions are not performed on a regular basis, but only in cases of stroke or other severe vaso-occlusive events, or when a patient has to be prepared for surgery. A minority of subjects are involved in chronic blood transfusion, which is used mostly to prevent cerebrovascular accidents but also in cases of cardiac, renal, or respiratory insufficiency. There is an increased prevalence of antierythrocytic alloimmunization in sickle cell patients, most probably because of the discrepancies in red cell antigens between mainly Caucasian blood donors and Afro-Caribbean recipients.

INDUCED HETERODUPLEX GENOTYPING OF TNF-α, IL-1β, IL-6 AND IL-10 POLYMORPHISMS ASSOCIATED WITH TRANSCRIPTIONAL REGULATION

We describe the construction and use of 7 induced heteroduplex generators, reagents for the rapid and unequivocal genotyping of nucleotide sequence polymorphism in TNF-α, IL-1β, IL-6 and IL-10. Polymorphisms detected are those previously associated with regulation of gene transcription: TNF-α positions −308 and −238; IL-1β position +3953; IL-6 position −174; and IL-10 positions −1082, −819 and −592. The reagents were used for analysis of allele and haplotype frequencies in a population of healthy Caucasian volunteer blood donors.

The DD genotype of the angiotensin-converting enzyme gene occurs in very low frequency in Australian Aboriginals.

The DD genotype of the angiotensin-converting enzyme (ACE) gene appears to be an independent risk factor for myocardial infarction, left ventricular hypertrophy and an increased incidence and rate of progression of renal disease. The high incidence of renal disease and end-stage renal failure in the Australian Aboriginal population has prompted investigation of ACE genotypes in these people. ACE genotypes were determined in four groups: (i) normal Australian Caucasian blood donors (n = 100), (ii) Caucasian renal transplant recipients (n = 173), (iii) normal Australian Aboriginals from a single tribe (n = 184), and (iv) Australian Aboriginals included in the renal-transplant programme (n = 94). The D allele frequency in the normal Australian Caucasian (54.5%) and renal transplant groups (57.2%) was similar. However, the D allele frequency in the normal Australian Aboriginal (3%) and Aboriginal renal patient group (14.4%) was significantly lower than both Caucasian groups. The D allele of the ACE gene has little or no influence on the renal disease of Australian Aboriginals.

Identification of a new DRB3*02 allele (DRB3*0207) by sequence-based typing.

A new DRB3*02 allele (DRB3*0207) was detected in a female Luxembourg Caucasian blood donor by sequence-based typing. The new allele differs from DRB3*0202 by two substitutions in codon 57 resulting in an amino acid change from a charged aspartic acid to a neutral valine. This is the first example of a DRB3 allele pair differing only at codon 57.

Phenol Sulfotransferase Pharmacogenetics in Humans: Association of CommonSULT1A1Alleles with TS PST Phenotype

The phenol sulfotransferases (PSTs) catalyze the sulfation of both small planar phenols and phenolic monoamines. Three highly homologous PST genes,SULT1A1, SULT1A2,andSULT1A3,are known to exist in humans. The prototypic biochemical phenotype associated with the enzyme encoded bySULT1A1is the thermal stable (TS) sulfation of 4 μM 4-nitrophenol (TS PST activity). Biochemical pharmacogenetic studies have demonstrated that individual variation in both TS PST activity and thermal stability in humans are inherited. As a step toward understanding molecular mechanisms responsible for the genetic regulation of PSTs in humans, we report here commonSULT1A1nucleotide polymorphisms that are associated with phenotypic variation in both platelet TS PST activity and thermal stability. When 905 human subjects were phenotyped for platelet TS PST activity and thermal stability, activity varied more than 50-fold, and thermal stability varied over 10-fold. DNA was isolated from the blood of 33 of these subjects selected on the basis of “extreme” TS PST phenotypes: high activity and high thermal stability; low activity and low thermal stability; or low activity and high thermal stability. These 33 subjects were genotyped forSULT1A1by PCR amplification and sequencing of the entire open reading frame (ORF) as well as approximately 1 kb of intron DNA sequence. One common allele,SULT1A1*2,was uniformly associated with both very low TS PST activity and low thermal stability. The allele frequency ofSULT1A1*2in a randomly selected population sample of 150 Caucasian blood donors was 0.31 (31%), indicating that approximately 9% of this population would be homozygous for that allele.

Analysis of HLA antigens in Caucasian patients with acute febrile neutrophilic dermatosis (Sweet’s syndrome)

MATERIAL AND METHODS Forty-one patients (30 females and 11 males; mean age 49 years [range, 9 to 75 years] with Sweet's syndrome according to standardized diagnostic criteria 2 and a control group of 500 geographically matched healthy Caucasian blood donors were studied. For HLA class I and class II typing a modified twostage complement microlymphocytotoxicity assay was used. HLA-DR analysis was performed in 24 patients with Sweet's syndrome and 286 controls by means of the microcytotoxicity method. 3 Appropriate statistical analysis was performed. 4-6 Odds ratios (ORs) were calculated 3,4 and Haldane's correction for the OR was used when appropriate. 5

Inherited Resistance to HIV-1 Conferred by an Inactivating Mutation in CC Chemokine Receptor 5: Studies in Populations with Contrasting Clinical Phenotypes, Defined Racial Background, and Quantified Risk

CC chemokine receptor 5 (CCR5) is a cell entry cofactor for macrophage-tropic isolates of human immunodeficiency virus-1 (HIV-1). Recently, an inactive CCR5 allele (designated here as CCR5-2) was identified that confers resistance to HIV-1 infection in homozygotes and slows the rate of progression to AIDS in heterozygotes. The reports conflict on the effect of heterozygous CCR5-2 on HIV-1 susceptibility, and race and risk levels have not yet been fully analyzed. Here we report our independent identification of CCR5-2 and test its effects on HIV-1 pathogenesis in individuals with contrasting clinical outcomes, defined race, and quantified risk. Mutant CCR5 alleles were sought by directed heteroduplex analysis of genomic DNA from random blood donors. Genotypic frequencies were then determined in (1) random blood donors from North America, Asia, and Africa; (2) HIV-1+ individuals; and (3) highly exposed-seronegative homosexuals with quantified risk. CCR5-2 was the only mutant allele found. It was common in Caucasians, less common in other North American racial groups, and not detected in West Africans or Tamil Indians. Homozygous CCR5-2 frequencies differed reciprocally in highly exposed-seronegative (4.5%, n = 111) and HIV-1-seropositive (0%, n = 614) Caucasians relative to Caucasian random blood donors (0.8%, n = 387). This difference was highly significant (p < 0.0001). By contrast, heterozygous CCR5-2 frequencies did not differ significantly in the same three groups (21.6, 22.6, and 21.7%, respectively). A 55% increase in the frequency of heterozygous CCR5-2 was observed in both of two cohorts of Caucasian homosexual male, long-term nonprogressors compared with other HIV-1+ Caucasian homosexuals (p = 0.006) and compared with Caucasian random blood donors. Moreover, Kaplan-Meier estimates indicated that CCR5-2 heterozygous seroconvertors had a 52.6% lower risk of developing AIDS than homozygous wild-type seroconvertors. The data suggest that homozygous CCR5-2 is an HIV-1 resistance factor in Caucasians with complete penetrance, and that heterozygous CCR5-2 slows the rate of disease progression in infected Caucasian homosexuals. Since the majority (approximately 96%) of highly exposed-seronegative individuals tested are not homozygous for CCR5-2, other resistance factors must exist. Since CCR5-2 homozygotes have no obvious clinical problems, CCR5 may be a good target for the development of novel antiretroviral therapy.

GM phenotypes influence the concentrations of the four subclasses of immunoglobulin G in normal human serum

The concentrations of the four subclasses of IgG were measured in the sera of 116 adult healthy Caucasian blood donors. Sera were also typed for nine GM determinants. All IgG subclass concentrations were significantly associated with GM phenotypes. The concentration of IgGl was lower in subjects with the GM 3 23 5,13 phenotype as compared with those who lacked this phenotype. Increased levels of IgG2 and IgG4 were associated with GM 1,2,3,17 23 5,13,21 and GM 1,3,17 23 5,13,21, respectively. IgG3 concentrations were the lowest for subjects with the GM 1,2,17 21 phenotype, intermediate for GM 1,3,17 5,13,21, and the highest in subjects with GM 3 23 5,13.

Haptoglobin polymorphism and chronic hepatitis C

Background/Aims: Haptoglobin (Hp) is a hemoglobin-binding acute phase protein characterized by a genetic polymorphism due to the existence of two different alleles encoding for the alpha chain of the protein. Three phenotypes have been described: Hp 1-1, Hp 2-1 and Hp 2-2. The latter two forms are known to possess immunoglobulin-like properties and play a role in the immune response. Recently, it has been shown that in subjects suffering from hepatitis C, serum Hp concentrations were lower than in the reference population. In the present study we examined whether the haptoglobin phenotype distribution in chronic HCV patients was different from the reference population. We also looked for possible relationships between Hp phenotypes and hepatitis C virus types and response to interferon α therapy. Moreover, Hp concentrations were determined. Methods: The study population consisted of 239 Caucasian patients with proven hepatitis C. Hp phenotypes were determined using starch gel electrophoresis of hemoglobin-supplemented serum, followed by peroxidase staining. Serum Hp concentrations were assayed with an immunonephelometric method. Hepatitis C virus was genotyped and classified according to an internationally accepted system. Two hundred and twenty healthy Caucasian blood-donors served as the reference population. Results: In the reference population, 35 individuals (15.9%) had Hp 1-1, 106 persons (48.2%) had Hp 2-1 and 79 had Hp 2-2 (35.9%), resulting in an Hp 1 allele frequency of 0.400, which is in agreement with the Hardy-Weinberg equilibrium. Hp phenotype distributions and Hp allele frequencies in the chronic hepatitis C virus patient group differed significantly from those obtained in the reference population. In the patient population, 59 individuals (24.7%) had Hp 1-1, 112 persons (46.9%) had Hp 2-1 and 68 had Hp 2-2 (28.5%). This resulted in an Hp 1 allele frequency of 0.481, which is in agreement with the Hardy-Weinberg equilibrium. No statistically significant differences were found between Hp phenotype distribution and hepatitis C virus types or response to interferon α therapy. Conclusions: The observed shift in Hp phenotype distribution in chronic hepatitis C may point to a role of Hp in the natural evolution of hepatitis C.

Serum IgG and IgG Subclass Contents in Different Gm Phenotypes

Different serum IgG and IgG subclass levels were found among Gm phenotypes of a normal population. One hundred and fifty-seven Caucasian blood donors were investigated for the reciprocal Gm allotypes on IgG subclass loci namely: for IgG1, G1m(f) and G1m(a); for IgG2, G2m(n) and G2m("); and for IgG3, G3m(b) and G3m(g), and subgrouped in the seven most common Gm phenotypes. The frequencies of Gm phenotypes and haplotypes were given, including numbers of the previously little known G2m(n,") heterozygous individuals. Mean serum quantities +/- SD and range of IgG, IgG1, IgG2, IgG3 and IgG4 were given for different Gm phenotypes. The IgG content was significantly lower in the Gm(f,",b/f,",b) phenotype in which the IgG2 levels were also significantly lower, compared with values of the other phenotypes. IgG3 levels were significantly lower in the Gm(a,",g/a,",g) phenotype compared with other phenotypes. These data imply the importance of Gm(f,",b/f,",b) and Gm(a,",g/a,",g) phenotypes causing lower amounts of IgG antibodies. In evaluating IgG subclass deficiency, the range for the low responding Gm(f,",b/f,",b) and Gm(a,",g/a,",g) phenotypes should be considered.

HLA–DR4 subtypes in new zealand polynesians. predominance of dw13 in the healthy population and association of dw15 with rheumatoid arthritis

To analyze HLA-DR4 alleles in New Zealand Polynesians with rheumatoid arthritis (RA). Thirty Polynesians and 30 Caucasians with RA, as well as 65 Polynesian and 60 Caucasian healthy blood donors, were DR4 subtyped using the polymerase chain reaction and sequence-specific oligonucleotide probes. The frequency of DR4 (DRB1*04) was increased in both Polynesian (P < 0.001) and Caucasian (P < 0.005) RA patients compared with race-matched controls. Dw4 (DRB1*0401) was detected in 15 of 30 Caucasian patients but only 2 of 30 Polynesian patients (P < 0.001). In Polynesians, RA was associated with Dw15 (DRB1*0405), which was present in 11 of 30 patients and 3 of 65 controls (P < 0.001). Dw13 (DRB1*0403) was the most frequent DR4 allele in healthy Polynesians, but was not significantly associated with RA. The predominance of the Dw13 subtype in Polynesians may explain in part the low prevalence of RA in this population. The association of Dw15 with RA in Polynesians supports the hypothesis that the third hypervariable region of DR beta determines susceptibility to RA.

Detection of 14 HLA-DQB1 alleles by oligotyping

Fourteen alleles have been identified by analysis of the nucleotide sequences encoding the HLA-DQB1 chain. This study describes the detection of these 14 alleles by selective gene amplification and sequence-specific hybridization with nonradioactive oligonucleotide probes. These techniques were employed to create an oligotyping system with two levels of resolution that provide versatility and the capacity for comprehensive detection of all polymorphic sequences. An initial low-resolution assay was employed to detect four major groups of alleles that are associated with the DQw1-w4 serological specificities. A further high-resolution assay was then employed to differentiate 14 individual DQB1 alleles. Appropriate control measures were also included to detect carry-over and to confirm hybridization specificity. This system was used to analyze the allele frequencies of DQB1-0301, -0302, and -0303 in 115 DQB1-03 ∗∗ Caucasian blood donors. Allele frequencies of oligotypes DQB1-05 ∗∗ and DQB1-06 ∗∗ were analyzed in 112 DQB1-01 ∗∗ Caucasian blood donors. This system was also utilized to identify oligotypes designated DQB1-0501 through DQB1-0503 and DQB1-0601 through DQB1-0605 in 35 Tenth International Histocompatibility Workshop DQw1 cell lines to examine the correlation with serological and cellular specificities. In one of these cell lines, an unexpected linkage was discovered between the DRB1 and DQB1 loci, suggesting a recombination event. Oligotyping is a precise and accurate method for directly defining polymorphic sequences and it promises to have a major impact on the future direction of HLA typing.

Comparison of IgM and IgG Anti‐A and Anti‐B Levels in Asian, Caucasian and Negro Donors in the North West Thames Region

This study was undertaken to test the widely held belief that higher levels of immune anti-A and anti-B are characteristic of Negro and Asian populations with a corresponding increased risk factor for AB0 haemolytic disease of the newborn. Overall, 300 serum samples from male and female Asian. Caucasian and Negro blood donors in the North West Thames Region of groups A, B and 0 were collected. The sera were titrated in microplates against pooled group A1 and pooled group B red blood cells. Although the results show that the highest levels for IgG anti-A and anti-B were found in group 0 female Negro donors, statistically these levels are not significantly higher than those of the other group 0 donors tested. We suggest that the potent anti-A and anti-B reported by others in Negro and Asian populations may arise from environmental rather than genetic factors.

Apolipoprotein A‐l/C‐lll gene cluster polymorphism in Saudi Arabians, Filipinos and Caucasians

We studied the polymorphic locus in the A-I/C-III gene cluster on the long arm of chromosome 11 in 147 Saudi Arabian, in 84 Filipino and in 69 Caucasian blood donors. Digestion of DNA yielded two fragments 4.2 kb and 3.2 kb long. The genotype distribution was the same in Arabs and Filipinos, but both were significantly different from Caucasians (p = 0.005 and 0.0005). The 3.2-kb allele occurred in 18% of the Saudi Arabians, in 23% of the Filipinos and in 4% of Caucasians. The frequency of the 3.2-kb allele was significantly higher in Arabs and Filipinos compared to Caucasians (p = 0.0005).