Estimation of haplotype frequency and linkage disequilibrium parameter in the HLA system.

Conventional formulae currently employed were extended to calculate the haplotype frequency and the corresponding variance for an arbitrary number (n) of loci when unrelated individuals were investigated. A formula is presented for the variance for the degree of three locus linkage disequilibrium.     

Linkage disequilibrium fine mapping and haplotype association analysis of the tau gene in progressive supranuclear palsy and corticobasal degeneration

Background: The haplotype H1 of the tau gene, MAPT, is highly associated with progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). Objective: To investigate the pathogenic basis of this association. Methods: Detailed linkage disequilibrium and common haplotype structure of MAPT were examined in 27 CEPH trios using validated HapMap genotype data for 24 single nucleotide polymorphisms (SNPs) spanning MAPT. Results: Multiple variants of the H1 haplotype were resolved, reflecting a far greater diversity of MAPT than can be explained by the H1 and H2 clades alone. Based on this, six haplotype tagging SNPs (htSNPs) that capture 95% of the common haplotype diversity were used to genotype well characterised PSP and CBD case–control cohorts. In addition to strong association with PSP and CBD of individual SNPs, two common haplotypes derived from these htSNPs were identified that are highly associated with PSP: the sole H2 derived haplotype was underrepresented and one of the common H1 derived haplotypes was highly associated, with a similar trend observed in CBD. There were powerful and highly significant associations with PSP and CBD of haplotypes formed by three H1 specific SNPs. This made it possible to define a candidate region of at least ∼56 kb, spanning sequences from upstream of MAPT exon 1 to intron 9. On the H1 haplotype background, these could harbour the pathogenic variants. Conclusions: The findings support the pathological evidence that underlying variations in MAPT could contribute to disease pathogenesis by subtle effects on gene expression and/or splicing. They also form the basis for the investigation of the possible genetic role of MAPT in Parkinson''s disease and other tauopathies, including Alzheimer''s disease.     

Importance of the renin-angiotensin system in the regulation of arterial blood pressure in conscious mice and rats

AIM: The present experiments were designed to determine the mechanism(s) for increased sensitivity to blockade of the renin-angiotensin system in mice in comparison with rats. METHODS: Mice and rats, with indwelling femoral arterial and venous catheters, were chronically administered angiotensin II or pharmacological inhibitors of the renin-angiotensin system as sodium intake was altered. RESULTS: Increasing sodium intake led to suppression of circulating renin, angiotensin II, and aldosterone in rats and mice in the absence of alterations in arterial blood pressure. Additional experiments demonstrated that continuous intravenous infusion of angiotensin II (20 ng kg(-1) min(-1)) significantly increased arterial blood pressure by approximately 35 mmHg in conscious rats at all levels of sodium intake (n = 6). In contrast, arterial pressure was unaffected by angiotensin II infusion in conscious mice under conditions of low sodium intake, although arterial pressure was increased by 16 mmHg when mice were administered a high sodium intake while infused with angiotensin II (n = 6). In comparison, blockade of the endogenous renin-angiotensin system led to significantly greater effects on arterial pressure in mice than rats. Continuous infusion of captopril (30 microg kg(-1) min(-1)) or losartan (100 microg kg(-1) min(-1)) resulted in a 55-90% greater fall in blood pressure in conscious mice in comparison with conscious rats. CONCLUSION: The present studies indicate that arterial pressure in mice is more dependent upon the endogenous renin-angiotensin system than it is in rats, but mice are more resistant to the hypertensive effects of exogenous angiotensin II.     

Linkage disequilibrium and haplotype analysis among ten single-nucleotide polymorphisms of interleukin 11 identified by sequencing of the gene

Interleukin 11 (IL11) is a member of the interleukin 6 (IL6)-related cytokine subfamily, which stimulates T cell-dependent development of immunoglobulin-producing B cells. IL11 is also an important paracrine regulator of bone metabolism that induces formation of osteoclasts. In the work reported here, we sequenced the entire IL11 structural gene of 48 alleles in a Japanese test population. These experiments identified ten single-nucleotide polymorphisms (SNPs) and determined their allelic frequencies. One polymorphism was identified upstream of exon 1, one in exon 3, four in intron 4 and four in the 3′ untranslated region (3′UTR) of exon 5. Based on the genotype data, we constructed six haplotypes in the tested population. Two-way comparisons of SNPs revealed two combinations in complete linkage disequilibrium, one with SNPs at nucleotide positions 2753, 3644, 5154, and 5568, and another with SNPs at positions 3686, 5141, and 5734. These results will be useful in disease-association studies where a contribution of the human IL11 gene has been suspected, especially in disorders affecting immune response and bone metabolism. Received: March 23, 2001 / Accepted: May 2, 2001     

Linkage disequilibrium analysis of two genes mapping on OFC3: PVR and PVRL2

Clefts of the lip with or without cleft palate (CL/P) are one of the most common birth defects, occurring in 1/700-1/1,000 infants born alive. The nature of the genetic contribution is still to be clarified; however, some chromosome regions and candidate genes have been proposed for this malformation. Recently, a couple of genes, PVR and PVRL2, mapping in the candidate region OFC3 on chromosome 19q13.31, have been investigated because of their homology to PVRL1, a gene previously shown to cause the Margarita Island CL/P-ectodermal dysplasia syndrome. In the present work, we investigated PVR and PVRL2 genes by family-based linkage disequilibrium analysis using a sample collected from the Italian population. In contrast to previous analyses on other populations, we could not find any statistically significant association between the markers alleles and non-syndromic clefting.     

Three-locus haplotype interactions in the analysis of linkage disequilibrium

We have shown that the application of Piazza's formula for the estimation of the "three-locus interaction delta" should not be used for that purpose. In addition, the method of Porta & McHugh, for the demonstration of haplotype interaction in associations between HLA and disease, leads to completely erroneous conclusions. Other methods of calculation that have been used to analyse haplotype interactions also appear to be based on erroneous concepts. We suggest that nothing more than the estimation of heterogeneity in simple 2 X 2 contingency tables should be used for the analysis of linkage disequilibria. This principle is applied to the HLA-A, -B and -C haplotype frequency tables of Baur & Danilovs. For the greater part of the HLA-B alleles, three-locus haplotype frequencies can be explained from the A, B and the B, C disequilibria, without any further haplotype interactions. The predominant exceptions in that respect are haplotypes containing the B44 allele, which has been shown to contain two subgroups and therefore, we are not justified to conclude that this exceptional behaviour of haplotypes with B44 should be attributed to three-locus haplotype interactions.     

Linkage disequilibrium and haplotype analysis among eight novel single-nucleotide polymorphisms in the human tissue-type plasminogen activator (t-PA) gene

Tissue-type plasminogen activator (t-PA), a serine protease, activates the conversion of plasminogen to the fibrinolytic protein, plasmin. The t-PA gene, mapped to chromosome 8p12-p11.2, contains 14 exons. An Alu insertion/deletion (I/D) polymorphism in this gene has been associated with an increased risk for myocardial infarction. In the work reported here we sequenced 11 kilobases (kb) of genomic DNA from 50 normal Japanese volunteers (100 alleles), to include all 14 exons of the t-PA gene, flanking intronic sequences, and 6 kb of the 5′ sequence. These experiments identified eight novel single-nucleotide polymorphisms (SNPs), in addition to the known Alu I/D polymorphism, from which genotypic data we constructed 12 haplotypes in the tested population. Two-way comparisons of SNPs and the Alu polymorphism revealed strong linkage disequilibrium between the Alu site and SNPs at positions 20,209 (χ² = 92.263) and 27,555 (χ² = 47.53), and between SNPs at positions 27,849 and 28,902 (χ² = 66.331). A phylogenic tree was constructed to infer a process of genome construction that would reflect the sequence variations we observed. Our results help to explain the lack of agreement among results of various disease-association studies in which a contribution of the human t-PA gene has been suspected but not always confirmed. Received: January 22, 2001 / Accepted: March 26, 2001     

Linkage disequilibrium and haplotype analysis among four novel single-nucleotide polymorphisms in the human leukemia inhibitory factor (LIF) gene

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine implicated in various pathological conditions, such as rheumatoid arthritis and osteoporosis. Despite the possible importance of LIF as a therapeutic target, little is known about the bioregulation of the human LIF gene. We here sequenced the entire structure of the LIF gene of 48 alleles in the Japanese population. These experiments identified four single-nucleotide polymorphisms (SNPs) and determined their allelic frequencies from a 48-allele sequence in the Japanese population. All four SNPs found in the LIF gene were located within exon 3, that is, a C/T at nucleotide (nt) position 3951, a C/G at nt position 4376, an A/C at nt position 4442, and a G/A at nt position 5961 (nucleotide numbering starts from the ATG start codon). Based on the genotypic data, we constructed four major haplotypes in the tested population. Two-way comparisons of SNPs revealed complete linkage disequilibrium between SNPs at positions 3951, 4376, and 4442. These results may prove to be useful as genetic markers for population-based disease-association studies in osteoporosis. Received: May 2, 2001 / Accepted: June 26, 2001     

Linkage disequilibrium and haplotype blocks in the MHC vary in an HLA haplotype specific manner assessed mainly by DRB1*03 and DRB1*04 haplotypes

First generation linkage disequilibrium (LD) and haplotype maps of the human major histocompatibility complex (MHC) have been generated in order to aid the unraveling of the numerous disease predisposing genes in this region by offering a first set of haplotype tagSNPs. Several parameters, like the population studied, the marker map used, the density of polymorphisms and the applied algorithm, are influencing the appearance of haplotype blocks and selection of tags. The MHC comprises a limited number of ancestral, conserved haplotypes. We address the impact of the underlying HLA haplotypes on the LD patterns, haplotype blocks and tag selection throughout the entire extended MHC (xMHC) by studying DR-DQ haplotypes, mainly those carrying DRB1*03 and DRB1*04 alleles. We observed significantly different degree and extent of LD calculated on different HLA backgrounds, as well as variation in the size and boundaries of the defined haplotype and tags selected. Our results demonstrate that the underlying ancestral HLA haplotypic architecture is yet another parameter to take into consideration when constructing LD maps of the xMHC. This may be essential for mapping of disease susceptibility genes since many diseases are associated with and map on particular HLA haplotypes.     

HLA-DQB1, -DQA1, -DRB1 linkage disequilibrium and haplotype diversity in a Mestizo population from Guadalajara, Mexico

HLA-DQB1, -DQA1, and -DRB1 genes were typed by polymerase chain reaction with sequence-specific primer (PCR-SSP) in 159 healthy volunteers from 32 families living in Guadalajara, Mexico. Three-locus genotype data from all family members were used to infer haplotypes in 54 unrelated individuals of the sample, from which estimate of segregating haplotype frequencies and linkage disequilibrium (LD) between loci were computed. Genotype distributions were concordant with Hardy-Weinberg expectations (HWE) for all three loci, and allele distributions were similar to the ones observed in other Latin-American populations. Of the 56 distinct three-site (DQB1-DQA1-DRB1) haplotypes observed in the sample, the five most common (i.e., with frequencies of five counts or more) were: *0302-*0301-*04, *0201-*0201-*07, *0301-*0501-*14, *0402-*0401-*08, and *0501-*0101-*01. These common three-locus haplotypes also contributed to the majority of the significant two-locus linkage disequilibria of these three sites.     

Linkage disequilibrium between S65C HFE mutation and HLA A29-B44 haplotype in Terceira Island,Azores

Our objective was to investigate the frequency of HFE gene mutations and to study linkage disequilibrium (LD) between HLA-Class I alleles and these mutations in the population of Terceira Island, Azores, Portugal. A total of 218 unrelated individuals were investigated. Three HFE mutations--C282Y, H63D, and S65C--were identified by restriction endonuclease digestion of polymerase chain reaction (PCR)-amplified genomic DNA. HLA-Class I alleles were typed by PCR-single-strand polymorphism. Gene frequencies and LD were estimated using Arlequin V 1.1. Six genotypes were found in the population: WT/WT (58.3%), H63D/WT (31.2%), H63D/H63D (2.3%), H63D/C282Y (0.9%), S65C/WT (4.1%), and C282Y/WT (3.2%). No cases of C282Y or S65C homozygosity were identified. HLA haplotype A3-B7 was in LD with C282Y; HLA alleles A29, B44, and HLA haplotype A29-B44 were in LD with S65C mutation. HFE gene frequencies in this population are similar to those in other European populations; HFE S65C mutation was found in LD with the alleles A29, B44, and with A29-B44 HLA haplotype.     

Linkage disequilibrium, haplotype and association studies of a chromosome 4 GABA receptor gene cluster: candidate gene variants for addictions.

Strong genetic contributions to individual differences in vulnerability to addictions are well supported by classical genetic studies. Linkage and association genome scans for addiction vulnerability have provided converging evidence for several chromosomal regions which are likely to harbor allelic variants that contribute to such vulnerability. We and others have delineated a candidate addiction-associated chromosome 4p12 "rSA3" region based on convergent data from association genome scanning studies in polysubstance abusers [Uhl et al. (2001); Am J Hum Genet 69(6):1290-1300], linkage-based studies in alcoholism [Long et al. (1998); Am J Med Genet 81(3):216-221; Reich et al. (1998); Am J Med Genet 81(3):207-215] and association-based studies for alcoholism and association-based studies for individual differences in electroencephalographic (EEG) spectral power phenotypes [Porjesz et al. (2002); Proc Natl Acad Sci USA 99(6):3729-3733; Edenberg et al. (2004); Am J Hum Genet 74(4):705-714]. The rSA3 region contains interesting candidate genes that encode the alpha 2, alpha 4, beta 1, and gamma 1 receptor subunits for the principal brain inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) [Covault et al. (2004); Am J Med Genet Part B 129B:104-109; Edenberg et al. (2004); Am J Hum Genet 74(4):705-714; Lappalainen et al. (2005); Alcohol Clin Exp Res 29(4):493-498]. We now report assessment of single nucleotide polymorphism (SNP) genotypes in this region in three samples of substance abusers and controls. These results delineate the haplotypes and patterns of linkage disequilibrium in this region, focus attention of the GABRA2 gene and identify modest associations between GABRA2 genotypes and addiction phenotypes. These results are consistent with modest roles for GABRA2 variants in addiction vulnerabilities.     

Comparison of family based haplotype methods using intragenic SNPs in candidate genes

The choice of an optimal marker strategy while analysing intragenic SNPs is presently of crucial importance, given the increasing amount of available data. Classical case/control association studies or family based association tests such as the TDT are very popular. However, as these methods are not able to analyse multiple markers simultaneously, different extensions have been proposed in order to use multiple markers. In the present study, the efficiency of five family based haplotypic methods to detect the role of candidate genes is evaluated and compared between them and with the classical single point TDT. Simulations of intragenic SNP maps are performed in recently founded populations. One or several SNPs are assumed to be the functional polymorphisms following different genetic models. Different modes of SNP combinations underlying the genetic susceptibility (epistasis or heterogeneity) are considered. Whereas haplotypic methods perform better in situations of heterogeneity, the TDT remains the most powerful approach in epistasis models as long as the marginal effect of one the SNPs involved in the susceptibility remains important. Haplotypic methods perform better than the TDT when the marginal effect of each SNP is small. Given the similar characteristics of intragenic LD in both old large populations and recently founded populations, in particular the weak correlation between LD and distance, our results are not likely to be specific to founder populations and can be generalized.     

Linkage disequilibrium on the COMT gene in French schizophrenics and controls

Catechol-O-methyltransferase (COMT) catalyzes the degradation of catecholamines and could therefore play a role in the etiology of schizophrenia. Moreover, microdeletions including the COMT locus have been found in schizophrenics presenting typical features of the velo-cardio-facial syndrome. In the present work, five single-strand conformation polymorphisms were detected in exons of the COMT gene. The linkage disequilibria between the polymorphisms were estimated, and the genotypic frequencies were calculated on a sample of 126 to 137 schizophrenics and 136 to 140 controls, depending on the marker. Patients and controls were matched for ethnicity and geographical origin. A trend toward association was found between schizophrenia and (i) genotype 11 of the Pml I polymorphism (p = 0.034; OR = 1.82); (ii) haplotype 1-2 for the Pml I and Bcl I polymorphisms (p = 0.022; OR = 1.75). The Pml I polymorphism is in complete linkage disequilibrium with the common Met→Val¹⁵⁸ substitution, which affects the activity of the enzyme. This finding suggests a possible minor effect of COMT in a multifactorial threshold model of vulnerability to schizophrenia. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 88:452–457, 1999. © 1999 Wiley-Liss, Inc.     

Linkage disequilibrium in isolated populations: Finland and a young sub-population of Kuusamo

Linkage disequilibrium (LD), non-random association of alleles at closely linked chromosomal loci, has been used as a tool in the identification of disease alleles, and this has led to an improved understanding of pathology in many monogenic Mendelian human diseases. We are currently moving from the mapping and identification of monogenic disease loci to attempts at identifying loci involved in predisposition to multifactorial diseases. In the selection of ascertainment strategies in the studies of these complex diseases, the extent of background LD in different populations is an important consideration. Here, we compare the extent of LD among the alleles of linked loci in a randomly ascertained sample of individuals from the Finnish population and a set of individuals ascertained from the region of Kuusamo, a small sub-population, founded some 13 generations ago, which has experienced very little subsequent immigration. Thirty-three microsatellite loci were genotyped in chromosomal regions on 13q, 19q, 21q, Xq, and Xp. The genetic diversity of these loci was determined separately in the general Finnish sample and in the Kuusamo sample. The X-chromosomal loci are characterised by higher levels of LD in the samples from Kuusamo than in the much larger (and older) general population of Finland, whereas in alleles of autosomal loci very little LD was seen in either of these two samples.