HLA class II allele frequencies in the Lebanese population

Aims

Being one of the most polymorphic genetic systems , the Human Leukocyte Antigen system is divided into class I (HLA-A, HLA-B and HLA-C) and class II (HLA-DP, -DQ and -DR). This study is the first and largest of its kind to describe the distribution of HLA-DQB1 and HLA-DRB1 alleles in Lebanon and the region.

Methods

Respectively, 560 and 563 Lebanese individuals referred for HLA typing and possible bone marrow/kidney donation were tested for HLA-DQB1 and HLA-DRB1 alleles using the polymerase chain reaction/sequence specific priming (PCR-SSP) method.

Results

Our data were compared to that of several populations with interesting common findings between the Lebanese, Jordanian, Bahraini, Saudi, Kuwaiti, Tunisian, Korean, Japanese, Thai, Irish, Bulgarian and Polish populations.

Conclusion

These data about the Lebanese population are going to aid future researchers to study the relation of HLA-DQB1 and HLA-DRB1 alleles with major and common diseases in the Lebanese population and will add to the available international literature associated with these loci. In addition it will serve as a reference for the future national bone marrow registry program in our country. We also reviewed the literature for the described association between HLA-DRB1 and -DQB1 loci and different disease entities.     

Virtual models of the HLA class I antigen processing pathway

Antigen recognition by cytotoxic CD8 T cells is dependent upon a number of critical steps in MHC class I antigen processing including proteosomal cleavage, TAP transport into the endoplasmic reticulum, and MHC class I binding. Based on extensive experimental data relating to each of these steps there is now the capacity to model individual antigen processing steps with a high degree of accuracy. This paper demonstrates the potential to bring together models of individual antigen processing steps, for example proteosome cleavage, TAP transport, and MHC binding, to build highly informative models of functional pathways. In particular, we demonstrate how an artificial neural network model of TAP transport was used to mine a HLA-binding database so as to identify HLA-binding peptides transported by TAP. This integrated model of antigen processing provided the unique insight that HLA class I alleles apparently constitute two separate classes: those that are TAP-efficient for peptide loading (HLA-B27, -A3, and -A24) and those that are TAP-inefficient (HLA-A2, -B7, and -B8). Hence, using this integrated model we were able to generate novel hypotheses regarding antigen processing, and these hypotheses are now capable of being tested experimentally. This model confirms the feasibility of constructing a virtual immune system, whereby each additional step in antigen processing is incorporated into a single modular model. Accurate models of antigen processing have implications for the study of basic immunology as well as for the design of peptide-based vaccines and other immunotherapies.     

Allele and haplotype frequencies of human leukocyte antigen-A, -B, -C,-DRB1, and -DQB1 in Chinese patients with hematological diseases

The human leukocyte antigen (HLA) system plays a central role in the immune response to pathogens, as well as in organ and allogenic hematopoietic stem cell transplantation (HSCT). Finding a five-locus (i.e., HLA-A, -B, -C, -DRB1, and -DQB1) matched unrelated donor for a patient awaiting HSCT is a major clinical challenge, due to the lack of HLA-identical sibling donors and the high polymorphism of HLA. To date, most studies providing HLA allele frequencies (AF) and haplotype frequencies (HF) in Chinese populations have focused on donors instead of the recipients and have provided data for three loci (HLA-A, -B, and -DR); however, data from five-locus HLA typing in a large sample of patients, especially those with hematological diseases, remains unavailable. Therefore, this study was designed to determine HLA AF and two-, three-, four- and five-locus HF in a large cohort of Chinese Han patients with hematological diseases. The AF and the HF were determined using high-resolution HLA typing data from 2,878 patients. The total number of HLA-A, -B, -C, -DRB1, and -DQB1 alleles was determined to be 48, 92, 49, 52, and 24, respectively. Hardy-Weinberg equilibrium (HWE) analyses indicated significant deviations from HWE for HLA-A, -C, -DRB1, and -DQB1 AF, but not for HLA-B locus. The three most common alleles at each locus were A*11:01, A*24:02, A*02:01; B*46:01, B*40:01, B*13:02; C*01:02, C*07:02, C*06:02; DRB1*09:01, DRB1*15:01, DRB1*07:01; DQB1*03:01, DQB1*03:03, and DQB1*06:01. Our data may help to determine whether the current bone marrow registry contains sufficient diversity to meet the demand.     

HLA-E is the ligand for the natural killer cell CD94/NKG2 receptors

CD94/NKG2 is a recently described receptor present on natural killer (NK) cells and certain T cells that is composed of the CD94 chain covalently associated with a member of the NKG2 family of molecules. Both chains are glycosylated members of the C-type lectin superfamily. The CD94/NKG2 receptors are functionally heterogenous depending on which NKG2 family member is associated with CD94. Initially, it was thought that CD94/NKG2 receptors recognized a broad array of HLA-A, -B and -C (classical), as well as the nonclassical HLA-G, MHC class I molecules. Instead, recent data have suggested that this receptor is specific for HLA-E complexed with a peptide derived from the signal sequence (residues 3–11) of certain classical MHC class I molecules. Position 2 (residue 4) in the signal sequence derived peptides appears pivotal in determining whether the HLA-E/peptide complex confers resistance to NK-mediated lysis. The potential roles that the CD94/NKG2-HLA-E receptor ligand interaction might play in infection and tumor development are discussed.     

Association and differentiation of MHC class I and II polymorphic Alu insertions and HLA-A, -B, -C and -DRB1 alleles in the Chinese Han population

In order to investigate the polymorphism of Alu insertions (POALINs) in the HLA region, we genotyped ten Alu loci (AluMICB, AluTF, AluHJ, AluHG, AluHF in the HLA class I region and AluDPB2, AluDQA2, AluDQA1, AluDRB1, AluORF10 in the HLA class II region) to determine their allele frequencies and associations with the HLA-A, HLA-B, HLA-C and HLA-DRB1 genes in the Chinese Han population. Our results showed the ten-loci POALINs varied in frequency between 0.003 and 0.425. By comparing the data of the ten-loci POALIN in Chinese Han with Japanese and Caucasian data, marked differences were observed between the three ethnic groups at the allelic or haplotypic levels. Each POALIN was in significant linkage disequilibrium with a variety of HLA-A, -B, -C and -DRB1 alleles, and was associated with a variety of HLA-A, -B, -C and -DRB1 allele in Chinese Han. This comparative study of multilocus POALINs in the HLA class I and II regions of the Chinese Han population shows that POALINs alone or as haplotypes together with the HLA class I and II alleles are informative genetic markers for the identification of HLA class I and II allele and variations, such as crossing over events within the same and/or different populations.     

Distribution of HLA class Ⅰ and class Ⅱ haplotypes in Chinese Han population based on family segregation

HLA haplotype analysis has important application value in human population genetics, anthropological research and HLA matching transplantation. Based on HLA-A, -B, -C, -DRB1 and -DQB1 genotyping data from 663 families including 663 leukemia patients and 991 related donors, the allele frequency (AF) and haplotype frequency (HF) of two-, three- and five-locus haplotype distribution patterns in the Chinese Han population were determined by family segregation. A total of 38 alleles at A locus, 75 alleles at B locus, 35 alleles at C locus, 53 alleles at DRB1 locus and 22 alleles at DQB1 locus were discovered in this population. The frequencies of these alleles were basically consistent with those of previous reports except for some tiny differences. The study found 11 A-C, 15 C-B, 4 B-DRB1 and 11 DRB1-DQB1 two-locus haplotypes with a frequency over 2%. The number of A-C-B and A-B-DRB1 three-locus haplotype with a frequency over 1% were 11 and 3 respectively. The most common HLA-A-C-B-DRB1-DQB1 haplotype (HF>1%) were A*3001-C*0602-B*1302-DR*0701-DQ*0202 (4.30%), A*0207-C*0102-B*4601-DR*0901-DQ*0303 (3.07%), A*3303-C*0302-B*5801-DR*0301-DQ*0201 (1.49%) and A*1101-C*0102-B*4601-DR*0901-DQ*0303 (1.01%). The results are helpful for finding matching donors for hematopoietic stem cell transplant patients and also contribute to transplant immunology, HLA-related diseases, research of human genetics and other fields.     

Comparison of HLA class I gene sequences. Derivation of locus-specific oligonucleotide probes specific for HLA-A, HLA-B, and HLA-C genes

The major histocompatibility complex in man contains at least 20 class I genes. Included within this family are three closely linked loci with 11-47 codominant alleles that encode the classical transplantation antigens HLA-A, -B, and -C. The study of individual HLA-A, -B, and -C genes is complicated both by the high degree of sequence homology among all members of the class I gene family and by the high degree of polymorphism exhibited by HLA-A, -B, and -C genes. Identification of potential locus-specific regions suitable for use as unique probes has been limited by the small number of nucleotide sequences available for comparison. In the present study, the nucleotide sequences of two cDNA clones, designated HLA-4 and HLA-10, that encode previously unsequenced alleles of HLA-C and HLA-A genes, respectively, are compared with those of other class I genes. From these intergenic and interallelic comparisons, it was deduced that the nucleotide sequence encoding amino acids 291-299 of the transmembrane region showed sufficient divergence between loci and similarity between alleles, to be suitable for the generation of locus-specific probes. Synthetic oligonucleotides were generated and shown to be highly locus-specific in hybridization. These probes were used successfully for the quantitation of the relative amounts of mRNA transcribed in human liver from HLA-A, -B, and -C genes; they should greatly simplify future studies of restriction fragment length polymorphisms of HLA-A, -B, and -C alleles as genetic markers of disease susceptibility.     

Amyloid precursor-like protein 2 association with HLA class I molecules

Amyloid precursor-like protein 2 (APLP2) is a ubiquitously expressed protein. The previously demonstrated functions for APLP2 include binding to the mouse major histocompatibility complex (MHC) class I molecule H-2K^d and down regulating its cell surface expression. In this study, we have investigated the interaction of APLP2 with the human leukocyte antigen (HLA) class I molecule in human tumor cell lines. APLP2 was readily detected in pancreatic, breast, and prostate tumor lines, although it was found only in very low amounts in lymphoma cell lines. In a pancreatic tumor cell line, HLA class I was extensively co-localized with APLP2 in vesicular compartments following endocytosis of HLA class I molecules. In pancreatic, breast, and prostate tumor lines, APLP2 was bound to the HLA class I molecule. APLP2 was found to bind to HLA-A24, and more strongly to HLA-A2. Increased expression of APLP2 resulted in reduced surface expression of HLA-A2 and HLA-A24. Overall, these studies demonstrate that APLP2 binds to the HLA class I molecule, co-localizes with it in intracellular vesicles, and reduces the level of HLA class I molecule cell surface expression.     

HLA determinants in an Australian population of hemochromatosis patients and their families.

The frequencies of different HLA-A and -B alleles in 77 Australian patients with hemochromatosis have been compared with frequencies of HLA alleles not associated with hemochromatosis in 63 of their heterozygous relatives and with published population frequencies. As for all other populations reported, an association of HLA-A3 and HLA-B7 with the disease was found. A weak association with HLA-B12 was also detected. No other significant positive or negative associations with HLA alleles were detected. In addition, HLA-A2 and -B12 were in significant linkage disequilibrium in patients but not in controls, which may indicate a new mutation or recent recombination between HLA-A and hemochromatosis either in our patient group or in the founding population. HLA-A1 and -B8 and HLA-A29 and -B12 were in linkage disequilibrium in controls but not in patients, suggesting that this population is not segregating a hemochromatosis allele on either of these haplotypes. Genetic linkage analysis using the program LIPED showed strong linkage in 23/24 families, most of which had additional HLA alleles (other than A3 and B7) associated with hemochromatosis. This provides evidence for a single hemochromatosis locus, possibly with more than one allele.     

HLA Class I and Class II Alleles and Haplotypes Confirm the Berber Origin of the Present Day Tunisian Population

In view of its distinct geographical location and relatively small area, Tunisia witnessed the presence of many civilizations and ethnic groups throughout history, thereby questioning the origin of present-day Tunisian population. We investigated HLA class I and class II gene profiles in Tunisians, and compared this profile with those of Mediterranean and Sub-Sahara African populations. A total of 376 unrelated Tunisian individuals of both genders were genotyped for HLA class I (A, B) and class II (DRB1, DQB1), using reverse dot-blot hybridization (PCR-SSO) method. Statistical analysis was performed using Arlequin software. Phylogenetic trees were constructed by DISPAN software, and correspondence analysis was carried out by VISTA software. One hundred fifty-three HLA alleles were identified in the studied sample, which comprised 41, 50, 40 and 22 alleles at HLA-A,-B,-DRB1 and -DQB1 loci, respectively. The most frequent alleles were HLA-A*02:01 (16.76%), HLA-B*44:02/03 (17.82%), HLA-DRB1*07:01 (19.02%), and HLA-DQB1*03:01 (17.95%). Four-locus haplotype analysis identified HLA-A*02:01-B*50:01-DRB1*07:01-DQB1*02:02 (2.2%) as the common haplotype in Tunisians. Compared to other nearby populations, Tunisians appear to be genetically related to Western Mediterranean population, in particular North Africans and Berbers. In conclusion, HLA genotype results indicate that Tunisians are related to present-day North Africans, Berbers and to Iberians, but not to Eastern Arabs (Palestinians, Jordanians and Lebanese). This suggests that the genetic contribution of Arab invasion of 7^th-11^th century A.D. had little impact of the North African gene pool.     

Apolipoprotein E Gene Polymorphism and Allele Frequencies in the Lebanese Population

Apolipoprotein E (ApoE) genotypes were studied in order to determine the prevalence in the Lebanese population and compare it with other populations. DNA from 160 unrelated healthy donors from our HLA-bank was used. ApoE genotype was determined using the CardioVascular Disease (CVD) StripAssay (this assay is based on a Polymerase Chain Reaction-Reverse Hybridization technique). The prevalence of genotypes E3/3, E3/4, and E2/3 was found to be 69%, 26%, and 22%, respectively, and 0.6% for each of E2/4 and E4/4 genotypes. The Lebanese population tested showed similarities to earlier reported ApoE genotypic distributions (high E3 allele frequency) but also peculiar differences especially to some Arabic countries (total absence of E2 allele among Saudis) and other populations. This is the first report from Lebanon that will serve as a template for future investigations of the prevalence of ApoE alleles in association with various clinical entities.     

Frequency distribution of the <Emphasis Type="Italic">G/A</Emphasis> alleles of the β-fibrinogen gene in the Lebanese population

Fibrinogen is a plasma protein that has been reported to be associated with an increased risk of atherothrombotic diseases and venous thrombosis. The most common polymorphism that has been studied so far in different populations is the G-455-->A polymorphism in the promoter region of the beta-fibrinogen gene. We studied 160 healthy unrelated Lebanese individuals for the prevalence of -455G/G, -455G/A and -455A/A genotypes of the beta-fibrinogen gene and the frequency of G and A alleles using a reverse hybridization PCR assay. The prevalence of the G/G, G/A, and A/A genotypes were found to be 60.6, 31.9 and 7.5%, respectively. The frequency of the G and A alleles were found to be 0.77 and 0.23, respectively. As compared to other ethnic groups, the Lebanese individuals were found to have a relatively high prevalence of the A allele which may predispose them to develop cardiovascular diseases as well as thrombotic events. This study provides additional unique genetic information pertaining to the Lebanese population.     

Detailed characterization of the peptide binding specificity of five common Patr class I MHC molecules

The chimpanzee (Pan troglodytes) is an important model for studying the immune response to several human pathogens, but the study of correlates of immunity has been hindered by the fact that little is known about the epitope-binding specificity of chimpanzee (Patr) class I MHC. In the present study we have characterized the peptide binding specificity of several common Patr class I molecules. Using single amino acid substitution analogs and large peptide libraries, quantitative peptide binding motifs have been derived for Patr A*0101, A*0701, A*0901, B*0101, and B*2401. Each molecule was found to bind peptides using position 2 and the C terminus as main anchor contacts. On the other hand, each Patr molecule is associated with a unique binding specificity, and the range of specificities is similar to that seen amongst HLA alleles. A high degree of cross-reactivity was noted between Patr A*0701 and Patr A*0901, suggesting the existence of a Patr-specific supertype. Consistent with previous studies suggesting that some cross-reactivity may exist between HLA and Patr alleles, Patr A*0901 was found to have an appreciable degree of cross-reactivity with molecules of the HLA A24-supertype. Finally, utilizing motif scans and peptide binding and intracellular cytokine staining assays, 77 hepatitis B virus (HBV)-derived epitopes were identified in five chimpanzees that were recently convalescent from acute HBV infection. Because the Patr alleles studied herein were found to be very common in two different chimpanzee populations, the present data should facilitate the use of chimpanzees for immunological studies.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

The polymorphisms of HLA class Ⅰ and Ⅱalleles were not associated with severe acute respiratory syndrome among recovered patients in Beijing: a case-control study

Severe acute respiratory syndrome (SARS) is a viral respiratory illness caused by a novel coronavirus (SARS-CoV), which emerged as a pandemic in 2003. The mechanism of the immune reaction initiated by SARS-CoV still remains unclear. Here we aimed to describe the genetic patterns of high-resolution HLA-A, -B, -C, -DRB1, and -DQB1, loci in recovered SARS patients from Beijing and examine the association between HLA genes and susceptibility or resistance to SARS. A total of 70 recovered Chinese Han SARS patients were recruited to donate convalescent plasma in 2003. HLA high-resolution typing was carried out using sequence based typing (SBT). Allele frequencies were calculated by direct counting, and were compared with the frequencies of HLA alleles of donors recruited by the China Marrow Donor Program between 2002 and 2015 using Fisher''s exact test. Significance of association was defined according to the Bonferroni method for multiple comparisons. We observed 20, 35, 21, 25, and 17 alleles respectively at HLA-A, -B, -C, -DRB1, and -DQB1 loci among the 70 recovered patients. We identified 12 alleles (HLA-A*02:10, -A*02:93, -A*03:02, -B*08:01, -B*15:152, -B*37:01, -DRB1*10:01, -DRB1*11:03, -DRB1*14:10, -DRB1*14:12, -DRB1*15:02, and -DQB1*05:10) showing a nominal association with SARS (P<0.05), but none remained significant after Bonferroni correction. The study suggests that high-resolution HLA alleles are unlikely to contribute significantly to the susceptibility or resistance to SARS-CoV infection in the northern Chinese population.     

Genetic analysis of HLA in the U.S. Schmiedenleut Hutterites.

The Hutterites are an Anabaptist population, highly inbred, with large family sizes and extensively documented pedigrees. As part of genetic-epidemiologic studies of the impact of HLA on fertility, HLA-A, -B, -C, -DR, and -DQ typing was performed on a total of 650 Schmiedenleut Hutterities in South Dakota. An extraordinary degree of homogeneity was found. HLA-A1, -A2, -A3, -A24, and -A26 accounted for 83%, HLA-B8, -B27, -B35, -B51, -Bw60, and -Bw62 for 75%, and HLA-DR1, -DR2, -DR3, and -DR4 for 66% of the antigens at the respective HLA-A, -B, and -DR loci. All Hutterites characterized for HLA were descendants of no more than 78 ancestors. However, family analysis identified only 45 unique HLA haplotypes thought to reflect the original gene pool. Eight haplotypes were particularly frequent, accounting for nearly 50% of all observed haplotypes; four of these were consistent with a European ancestry. Coefficients measuring linkage disequilibrium were computed from haplotypes identified by family analysis. Overall, HLA analysis portrayed the Schmiedenleut Hutterities as a homogeneous and unique population, with disequilibrium among particular alleles and a spectrum of common and uncommon European haplotypes.     

Polymorphism of HLA class I loci HLA-A, -B, -C, in the Mandenka population from eastern Senegal]

A sample of 162 Mandenkalu from Eastern Senegal has been typed for three HLA class I loci: HLA-A, -B and -C. The Mandenka population presents a very high genetic variability with 15 alleles for locus A, 24 alleles for locus B, and at least 8 alleles for locus C. The calculated heterozygosities for the three loci A, B, and C are respectively 0.884, 0.944 and 0.829. The Mandenkalu allelic frequencies are close to that found in other sub-Saharan populations. They show, however, some peculiarities like the occurrence of the Bw 56 allele and the high frequencies of both B5 and B35.     

Sequence variability analysis of human class I and class II MHC molecules: functional and structural correlates of amino acid polymorphisms

Major histocompatibility complex class I (MHCI) and class II (MHCII) molecules display peptides on antigen-presenting cell surfaces for subsequent T-cell recognition. Within the human population, allelic variation among the classical MHCI and II gene products is the basis for differential peptide binding, thymic repertoire bias and allograft rejection. While available 3D structural analysis suggests that polymorphisms are found primarily within the peptide-binding site, a broader informatic approach pinpointing functional polymorphisms relevant for immune recognition is currently lacking. To this end, we have now analyzed known human class I (774) and class II (485) alleles at each amino acid position using a variability metric (V). Polymorphisms (V>1) have been identified in residues that contact the peptide and/or T-cell receptor (TCR). Using sequence logos to investigate TCR contact sites on HLA molecules, we have identified conserved MHCI residues distinct from those of conserved MHCII residues. In addition, specific class II (HLA-DP, -DQ, -DR) and class I (HLA-A, -B, -C) contacts for TCR binding are revealed. We discuss these findings in the context of TCR restriction and alloreactivity.     

Ancestral association between HLA and HFE H63D and C282Y gene mutations from northwest Colombia

A significant association between HFE gene mutations and the HLA-A*03-B*07 and HLA-A*29-B*44 haplotypes has been reported in the Spanish population. It has been proposed that these mutations are probably connected with Celtic and North African ancestry, respectively. We aimed to find the possible ancestral association between HLA alleles and haplotypes associated with the HFE gene (C282Y and H63D) mutations in 214 subjects from Antioquia, Colombia. These were 18 individuals with presumed hereditary hemochromatosis (“HH”) and 196 controls. The HLA-B*07 allele was in linkage disequilibrium (LD) with C282Y, while HLA-A*23, A*29, HLA-B*44, and B*49 were in LD with H63D. Altogether, our results show that, although the H63D mutation is more common in the Antioquia population, it is not associated with any particular HLA haplotype, whereas the C282Y mutation is associated with HLA-A*03-B*07, this supporting a northern Spaniard ancestry.     

Five-locus HLA typing of hematopoietic stem cell donor volunteers using PCR sequence specific primers

We have developed a strategy for five-locus human leukocyte antigen (HLA) typing of hematopoeitic stem cell (HSC) donors using the polymerase chain reaction with sequence-specific primers (PCR-SSP). The PCR-SSP method is robust, reproducible, and accurate. New PCR-SSP mixtures can be added as required and all reactions are carried out under the same conditions, which can easily be applied to the typing of other loci, e.g., ABO blood groups. Initially, 127 PCR-SSP reactions were used to detect simultaneously HLA-A, -B, -C, -DRB1/3/4/5, and DQB1 alleles, differentiated generally to the level of the first two digits of the allele name, essentially equivalent to the serological split specificity. Approximately 40% of subjects were tested against a further 29 HLA-A, -B SSP mixtures to exclude rare alleles and unambiguously assign a two-digit HLA allele family. This gave an overall typing resolution equivalent to or greater than the split specificity level and covered all HLA-A, -B, -C, -DRBland DQB1 alleles listed in the WHO's Nomenclature for Factors of the HLA System, 2000. The Welsh Bone Marrow Donor Registry has used this strategy to HLA type over 35,000 HSC donors over 9 years. Comprehensive and accurate five-locus HLA typing allows confident and rapid identification of potential matched HSC donors for patients requiring stem cell transplantation generally without the need for typing additional loci. This allows resources to be focused directly on allele level typing of DRB1 and other loci. This strategy decreases overall donor work-up time, which is a major benefit to patients.     

Factor XIII gene <Emphasis Type="Italic">V34L</Emphasis> mutation in the Lebanese population: Another unique feature in this community?

We studied the distribution of the Factor XIII gene V34L polymorphism in a sample of healthy Lebanese individuals to assess its prevalence and compare it with other populations. Factor XIII genotypes were determined using the Cardiovascular Disease (CVD) StripAssay (ViennaLab, Austria), which is based on a Polymerase Chain Reaction-Reverse hybridization technique. DNA from 205 unrelated healthy donors from our HLA database was used. The prevalence of Wild type, heterozygous, and homozygous genotypes was found to be 74.2%, 22.4%, and 3.4% respectively. The sampled Lebanese population showed that the prevalence of V34L carriers (25.8%) was lower than Caucasians in general (44.3%) and, interestingly, with a low allele frequency of 0.14 similar to that in Blacks and South Asians. This first report from Lebanon sheds light on an additional unique genetic feature of this population and will prospectively serve as a baseline statistical data for future investigations of the prevalence of Factor XIII V34L mutation in association with various clinical entities notably cardiovascular diseases.