Trans-ethnic fine mapping of a quantitative trait locus for circulating angiotensin I-converting enzyme (ACE)

Circulating angiotensin I-converting enzyme (ACE) levels are influenced by a major quantitative trait locus (QTL) that maps to the ACE gene. Phylogenetic and measured haplotype analyses have suggested that the ACE-linked QTL lies downstream of a putative ancestral breakpoint located near to position 6435. However, strong linkage disequilibrium between markers in the 3' portion of the gene has prevented further resolution of the QTL in Caucasian subjects. We have examined 10 ACE gene polymorphisms in Afro-Caribbean families recruited in JAMAICA: Variance components analyses showed strong evidence of linkage and association to circulating ACE levels. When the linkage results were contrasted with those from a set of British Caucasian families, there was no evidence for heterogeneity between the samples. However, patterns of allelic association between the markers and circulating ACE levels differed significantly in the two data sets. In the British families, three markers [G2215A, Alu insertion/deletion and G2350A] were in complete disequilibrium with the ACE-linked QTL. In the Jamaican families, only marker G2350A showed strong but incomplete disequilibrium with the ACE-linked QTL. These results suggest that additional unobserved polymorphisms have an effect on circulating ACE levels in Jamaican families. Furthermore, our results show that a variance components approach combined with structured, quantitative comparisons between families from different ethnic groups may be a useful strategy for helping to determine which, if any, variants in a small genomic region directly influence a quantitative trait.     

Angiotensin-1-converting enzyme (ACE) plasma concentration is influenced by multiple ACE-linked quantitative trait nucleotides

Circulating angiotensin-1-converting enzyme (ACE) is a highly heritable trait, and a major component of the genetic variance maps to the region of the ACE gene. The strong effect of the locus, and the interest in ACE as a candidate gene for cardiovascular disorders, has led to extensive investigation of its relationship to the ACE phenotype, providing one of the most complete examples of quantitative trait locus (QTL) analysis in humans. Resequencing of ACE followed by haplotype analysis in families of British and French origin has shown that the genetic variants that are primarily associated with the ACE trait map to an 18 kb interval flanked by two intragenic, ancestral recombination breakpoints. This critical interval contains dozens of ACE-associated variants in Caucasians, but identification of which of these directly influence ACE concentration is ambiguous because of the almost complete linkage disequilibrium in European populations. In a complementary sequencing and genotyping study of individuals from West African families, we show that this population has much greater haplotype diversity across the gene. Through analysis of the contrasting relationships of the trait phenotype with haplotypes that carry different allelic combinations from those observed in Caucasians, we demonstrate that (at least) two major intragenic sites within the critical interval and (at least) one minor promoter site are associated with the ACE quantitative trait through additive effects. These results point to the importance of analysing diverse populations with different gene genealogies in gene-association studies.     

High-resolution genetic mapping of the ACE-linked QTL influencing circulating ACE activity

Fine-mapping of trait loci through combined linkage and association analysis is an important component of strategies designed to identify causative gene variants, particularly in situations where the trait may be influenced by one or more of many polymorphisms within the same gene. Angiotensin-1 converting enzyme (ACE) provides one of the best models for developing and testing such methodologies, as a major fraction of the heritable variation in the activity of the angiotensin-1 converting enzyme (ACE) is tightly linked to the ACE gene. Moreover, ACE contains many frequent polymorphisms that are in strong linkage disequilibrium with each other. Although none of these variants induces a significant amino-acid change, one or more, either singly or in combination, are likely to have a strong effect on the quantitative phenotype. Here, we show that measured-haplotype analysis of SNP data from a large European family cohort can be used to localise the major ACE-linked genetic factors influencing the trait to a 16 kb interval within the gene, thus limiting the number of ACE variants that need to be considered in future studies designed to elucidate their biological effects. The approaches developed will be applicable to the fine-mapping of other quantitative trait loci in humans.     

Haplotypes in the APOA1-C3-A4-A5 gene cluster affect plasma lipids in both humans and baboons

Genetic studies in non-human primates serve as a potential strategy for identifying genomic intervals where polymorphisms impact upon human disease-related phenotypes. It remains unclear, however, whether independently arising polymorphisms in orthologous regions of non-human primates leads to similar variation in a quantitative trait found in both species. To explore this paradigm, we studied a baboon apolipoprotein gene cluster (APOA1/C3/A4/A5) for which the human gene orthologs have well-established roles in influencing plasma HDL-cholesterol and triglyceride concentrations. Our extensive polymorphism analysis of this 68 kb gene cluster in 96 pedigreed baboons identified several haplotype blocks each with limited diversity, consistent with haplotype findings in humans. To determine whether baboons, like humans, also have particular haplotypes associated with lipid phenotypes, we genotyped 634 well-characterized baboons using 16 haplotype tagging SNPs. Genetic analysis of single SNPs, as well as haplotypes, revealed an association of APOA5 and APOC3 variants with HDL-cholesterol and triglyceride concentrations, respectively. Thus, independent variation in orthologous genomic intervals does associate with similar quantitative lipid traits in both species, supporting the possibility of uncovering human quantitative trait loci genes in a highly controlled non-human primate model.     

Characterization of polymorphisms in the promoter of the human angiotensin II subtype 1 (AT1) receptor gene

In this study eight sequence variants in the functional promoter of the human angiotensin II subtype 1 (AT1 or AGTR1) receptor gene are reported. Six of these variants are in nearly total linkage disequilibrium with each other and occur with a frequency of 15.7%. By haplotype estimation this group of eight sequence variants is characterized by only five haplotypes. There is no linkage disequilibrium between one of these haplotypes and the AT1+1166A/C variant. The finding of polymorphic sites in the functional promoter of the human AT1 locus will be beneficial to the study of the role of the AT1 receptor gene in hypertension and other cardiovascular diseases.     

No contribution of angiotensin-converting enzyme (ACE) gene variants to severe obesity: a model for comprehensive case/control and quantitative cladistic analysis of ACE in human diseases

Candidate gene analyses are often inconclusive owing to genetic or phenotypic heterogeneity, low statistical power, selection of nonfunctional SNPs, and inadequate statistical analysis of the genetic architecture. Angiotensin-converting enzyme (ACE) is involved in adipocyte growth and function and the ACE-processed angiotensin II inhibits adipocyte differentiation. Associations between body mass index (BMI) and ACE polymorphisms have been reported in general populations, but the contribution to severe obesity of this gene, which is located under an obesity genome-scan linkage peak on 17q23, is unknown. ACE is one of the most studied genes and markers responsible for variation in circulating ACE enzyme levels have been extensively characterised. Eight of these variants were genotyped in 1054 severely obese cases and 918 nonobese controls, as well as 116 nuclear families from the genome scan (n=447), enabling the known clades to be inferred. Qualitative analysis of individual single-nucleotide polymorphisms (SNPs), haplotypes, clades, and diploclades demonstrated no significant associations (P<0.05) after minimal correction for multiple testing. Quantitative analysis of clades and diploclades for BMI, waist-to-hip ratio, or ZBMI in children were also not significant. This rigorous, large-scale study of common, well-defined, severe polygenic obesity provides strong evidence that functionally relevant sequence variation in ACE, whether it is defined at the level of SNPs, haplotypes, or clades, is not associated with severe obesity in French Caucasians. Such a study design exemplifies the strategy needed to clearly define the contribution of the ACE gene to the plethora of complex genetic diseases where weak associations have been previously reported.     

Association of angiotensin-converting enzyme polymorphisms with systemic lupus erythematosus and nephritis: analysis of 644 SLE families

Angiotensin II is a strong candidate for the perpetuation of autoimmunity, nephritis and visceral damage in systemic lupus erythematosus (SLE). Our goal was to determine whether angiotensin-converting enzyme (ACE) gene polymorphisms are associated with SLE and/or lupus nephritis (LN). We genotyped 644 SLE patients and 1130 family members for three ACE gene polymorphisms: Alu insertion/deletion (I/D), 23949 (CT)(2/3) and 10698 (G)(3/4). All patients met the American College of Rheumatology (ACR) criteria for SLE, and all LN patients met ACR renal criteria and/or had biopsy evidence of LN. We used the transmission/disequilibrium test (TDT) to examine associations between each polymorphism and SLE, including Caucasian, non-Caucasian, and LN subgroups. We also examined transmission of haplotypes defined by these polymorphisms. The ACE I/D polymorphism was associated with SLE among non-Caucasians (61% transmission, P = 0.026) and the 23949 (CT)(2/3) polymorphism was associated with LN among non-Caucasians (69% transmission, P = 0.014). Several haplotypes defined by these 2 markers demonstrated strikingly increased transmission among non-Caucasians (81% - 66% transmission, P = 0.0046 to 0.010). Due to the choice of study design and analytic method these results are unlikely to be due to population admixture. Our findings suggest that DNA sequence variation in the ACE gene influences the risk of developing SLE and LN.     

Measured haplotype analysis of the aldosterone synthase gene and heart size

Gene-association studies of heart size and the aldosterone synthase (CYP11B2) gene have produced inconsistent results, possibly because of limitations in the sample size and/or the number and location of the polymorphisms. An analysis of six polymorphisms spanning 6 kb of the CYP11B2 gene in Caucasian British families revealed a limited number of haplotypes because of strong linkage disequilibrium over this small region. The genotype and haplotype information was used in an association study involving 955 members of 229 families phenotyped for echocardiographic measures of heart size. In a mixed effects linear modelling analysis, the G5937C polymorphism was associated with cardiac wall thickness (P=0.02), and the intron conversion and A4550C polymorphisms were associated with left ventricular cavity size (P=0.02 and 0.002, respectively). Measured haplotype analyses confirmed the association of alleles at the intron conversion and G5937C polymorphisms with cardiac wall thickness (P=0.02), and alleles at the intron conversion polymorphism with left ventricular cavity size (P=0.04). The polymorphisms contributed to 2.0-3.4% of the variability in these traits. In summary, genetic polymorphisms at the CYP11B2 gene make a small contribution to quantitative variation in echocardiographic measures of heart size. These results point to the importance of analysing the full extent of genetic variation that captures the haplotype structure of a locus in gene association studies.     

浙江地区汉族人群caspase-8、-10基因四个单核苷酸多态性位点的单倍型研究

目的分析汉族人群caspase-8、-10基因的单核苷酸多态性（single nucleotide polymorphisins，SNPs）位点及其构成的单倍型，为研究caspase-8、-10基因与多基因复杂疾病的关联分析奠定基础。方法采用PCR、变性高效液相色谱技术和DNA测序技术检测caspase-10基因的第2．5外显子，caspase-8基因的第8-10外显子及其部分侧翼序列的多态性位点；分析配对位点的连锁不平衡关系，最大期望值法估算它们构成的单倍型。结果（1）caspase-10基因的第2、5外显子分别检出一个SNP位点A2823G和A12799G，其中A12799G是新发现的低信息度的SNP；caspase-8基因中检测到3个SNP位点A43466G、G51484A、G52951A，分别位于第8、9外显子和第9内含子；它们均未改变所编码蛋白的一级结构；（2）caspase-10基因中A2823G位点与caspase-8基因中3个位点间已达到连锁平衡，caspase-8基因中A43466G与G52951A、G51484A与G52951A也达到连锁平衡，连锁不平衡系数分别接近于0；只有A43466G与G51484A存在强的连锁不平衡，连锁不平衡系数接近于1；（3）caspase-10基因的A2823G位点与caspase-8基因的3个位点预计产生11种单倍型，其中A-2823／A-43466／G-51484／G-52951是主要单倍型，频率为0．381l，其次是A．2823／A-43466／G-51484／A-52951，频率为0．2536；这4个SNP位点联用，多态信息含量可达到0．7106。结论浙江地区汉族人群caspase-10、-8基因中的SNP位点至少处于3个不同的单倍型块；联合多个相邻的位点构成单倍型，可以弥补单个SNP信息度较低的不足。     

Lack of significant association between -1021C-->T polymorphism in the dopamine beta hydroxylase gene and attention deficit hyperactivity disorder

Recent trends in medications for attention deficit hyperactivity disorder (ADHD) suggest that norepinephrine (NE) deficiency may contribute to the disease etiology. Dopamine beta hydroxylase (DBH) is the key enzyme which converts dopamine to NE and since DBH gene is considered a major quantitative trait locus for plasma DBH activity, genetic polymorphism may lead to altered NE neurotransmission. Several polymorphisms including a 5' flanking -1021C-->T polymorphism, was reported to be associated with changed DBH activity and an association between -1021C-->T polymorphism with ADHD was observed in Han Chinese children. We have carried out family-based studies with three polymorphisms in the DBH gene, -1021C-->T polymorphism, exon 2*444g/a and intron 5 TaqI RFLP, to explore their association with Indian ADHD cases. Allele and genotype frequency of these polymorphisms in ADHD cases were compared with that of their parents and a control group. Haplotypes obtained were analyzed for linkage disequilibrium (LD). Haplotype-based haplotype relative risk analysis and transmission disequilibrium test showed lack of significant association between transmission of the polymorphisms and ADHD. A haplotype comprising of allele 1 of all polymorphisms showed a slight positive trend towards transmission from parents to ADHD probands. Strong LD was observed between *444g/a and TaqI RFLP in all the groups. However, low D' values and corresponding log of odds scores in the control group as compared to the ADHD families indicated that, the incidence of the two polymorphisms being transmitted together could be higher in ADHD families.     

Protein kinase C-beta 1 gene variants are not associated with autism in the Irish population

Some evidences indicate that protein kinase C-beta 1 (PRKCB1) gene may be a predisposition locus of autism. A recent study reported evidence of association between autism and two haplotypes made up of six noncoding single nucleotide polymorphisms in the PRKCB1. To attempt replication of their findings, we examined the same six single nucleotide polymorphisms of PRKCB1 in 171 Irish autism trios. The haploview program was used to calculate D' as a measure of linkage disequilibrium. The transmission disequilibrium test for single nucleotide polymorphism markers and haplotypes was carried out using the TDTPHASE and PDTPHASE from the UNPHASED version 2.404 programs. Transmission disequilibrium test analysis showed no evidence of association for any of the six single nucleotide polymorphisms at the PRKCB1 that we studied, or any of their haplotypes. Our data do not support the finding that the PRKCB1 gene variants contribute risk for the development of autism.     

海南汉族人群中α和β纤维蛋白原基因核苷酸多态性及其单倍型与缺血性脑卒中的关联分析

目的研究α纤维蛋白原基因的Taq Ⅰ多态性和β纤维蛋白原基因-455G／A、-249C／T、-148 C／T、＋1689T／G、βBsmA ⅠG／C、448G／A、Be／ⅠG／A、Hinf Ⅰ A／C单核苷酸多态性及其单倍型与缺血性脑卒中的关系。方法用比浊法测定160例海南籍缺血性脑卒中和130名海南籍对照个体的血浆纤维蛋白原浓度，用PCR-限制性片段长度多态法确定基因型。用EH＋程序分析核苷酸多态性的连锁不平衡关系及单倍型，用卡方检验分析病例组和对照组的等位基因频率、基因型频率及单倍型频率的差异。结果-455G／A、-148C／T、448G／A多态性的基因型频率、等位基因频率在病例组和对照组之间的差异有统计学意义（P〈0．01），其余6个核苷酸多态性的基因型频率、等位基因频率在病例和对照组间的差异无统计学意义（P〉0．05），A^-455、T^-148、A^448携带者患缺血性脑卒中的相对危险度比非携带者分别大2．46倍、2．30倍和2．08倍。连锁不平衡分析未发现所分析的区域内存在单倍型板块。9个位点构建的单倍型在病例组和对照组之间的差异无统计学意义，以4个位点构建的单倍型中，某些单倍型在病例组和对照组之间的差异有统计学意义，对照组中某些携带G^-455、C^148、G^448位点的单倍型的频率高于病例组，而病例组中某些携带A^-455、T^-148、A^448位点的单倍型的频率高于对照组。结论多个位点和单倍型分析的结果提示8纤维白原455G／A、-148C／T、448G／A可能是海南汉族人群中与缺血性脑卒中关联的危险因素。     

Linkage study of two polymorphisms at the dopamine D3 receptor gene and attention-deficit hyperactivity disorder

Data from animal studies suggest that the dopamine D3 receptor gene may have a role in locomotion and behavioral regulation. Therefore, this gene has been suggested as a candidate for attention-deficit hyperactivity disorder (ADHD). The dopamine D3 receptor gene (DRD3) has two common polymorphisms, one in exon I that changes a Serine to Glycine (Ser9Gly) and alters the recognition site for the restriction enzyme MscI [Lannfelt et al., 1992]. The other common polymorphism is located in intron 5 and results in the change of a restriction site for MspI [Griffon et al., 1996]. We investigated the possibility of linkage of the dopamine D3 receptor gene in 100 small, nuclear families consisting of a proband with ADHD, their parents, and affected siblings. We examined the transmission of the alleles of each of these polymorphisms and the haplotypes of both polymorphisms using the transmission disequilibrium test [Spielman et al., 1993]. We did not observe biased transmission of the alleles at either polymorphism or any haplotype. Our findings using this particular sample do not support the role of the dopamine D3 gene in ADHD. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:114-117, 2000.     

Lipoprotein lipase variants associated with an endophenotype of hypertension: hypertension combined with elevated triglycerides

Previously, we observed that young-onset hypertension was independently associated with elevated plasma triglyceride(s) (TG) levels to a greater extent than other metabolic risk factors. Thus, focusing on the endophenotype--hypertension combined with elevated TG--we designed a family-based haplotype association study to explore its genetic connection with novel genetic variants of lipoprotein lipase gene (LPL), which encodes a major lipid metabolizing enzyme. Young-onset hypertension probands and their families were recruited, numbering 1,002 individuals from 345 families. Single-nucleotide polymorphism discovery for LPL, linkage disequilibrium (LD) analysis, transmission disequilibrium tests (TDT), bin construction, haplotype TDT association and logistic regression analysis were performed. We found that the CC- haplotype (i) spanning from intron 2 to intron 4 and the ACATT haplotype (ii) spanning from intron 5 to intron 6 were significantly associated with hypertension-related phenotypes: hypertension (ii, P=0.05), elevated TG (i, P=0.01), and hypertension combined with elevated TG (i, P=0.001; ii, P<0.0001), according to TDT. The risk of this hypertension subtype increased with the number of risk haplotypes in the two loci, using logistic regression model after adjusting within-family correlation. The relationships between LPL variants and hypertension-related disorders were also confirmed by an independent association study. Finally, we showed a trend that individuals with homozygous risk haplotypes had decreased LPL expression after a fatty meal, as opposed to those with protective haplotypes. In conclusion, this study strongly suggests that two LPL intronic variants may be associated with development of the hypertension endophenotype with elevated TG.     

Genetic association between Notch4 polymorphisms and Japanese schizophrenics

The objective of this study was to investigate whether three single nucleotide polymorphisms of the Notch4 gene are associated with the onset of schizophrenia. To confirm the linkage disequilibrium among these three single nucleotide polymorphisms of the gene, the three single nucleotide polymorphisms were genotyped by a polymerase chain reaction-restriction-fragment length polymorphism method for all samples. The genotypic frequencies of each single nucleotide polymorphism in the schizophrenic were compared with respective controls using a chi method. To check linkage disequilibrium, the haplotype frequency program was utilized. No statistical association between the two single nucleotide polymorphisms of the Notch4 gene and schizophrenia was observed in our Japanese samples. Although one nonsynonymous single nucleotide polymorphism did show a weakly significant P-value, its allelic frequencies are not positive. Two of the single nucleotide polymorphisms showed strong linkage disequilibrium in our Japanese samples. The single nucleotide polymorphism between the other two single nucleotide polymorphisms showed a weaker linkage disequilibrium with the others. Our study suggests that the three single nucleotide polymorphisms are not associated with the onset of schizophrenia. The linkage disequilibrium of this locus indicates that there is genetic heterogeneity in the Notch4 gene. Linkage disequilibrium may differ among ethnic groups, and so a larger study should be performed in this region.     

Mapping quantitative effects of oligogenes by allelic association

Regression analysis of a quantitative trait as a function of a single diallelic polymorphism has been extended to allelic association by composite likelihood under the Malecot model for multiple markers. We applied the method to 10 single nucleotide polymorphisms (SNPs) spanning 27 kb of the angiotensin-I converting enzyme ( ACE ) gene in British families, localising a causal SNP between G2530A and 4656(CT)3/2 in the 3' region, at a distance of 21.6±0.9 kb from the most proximal SNP T-5491C. Neither they nor the I / D polymorphism is causal. To clarify genetic parameters we applied combined segregation, linkage and association analysis. Stronger evidence for the 3' region was obtained, with significant evidence of a lesser 5' effect as reported in French and Nigerian families. However, rigorous confirmation requires that the causal SNPs be identified. Both Malecot and parametric analysis appear to have high power by comparison with alternative methods for localizing oligogenes and their causal polymorphisms.     

血管紧张素原基因单倍型与原发性高血压的关联研究

目的 研究中国人血管紧张素原（angiotensinogen,AGT)基因多态性及单倍型与原发性高血压的关系。方法 在335例高血压患者与196名血压正常人中采用PCR－限制性酶切片段长度多态性法检测血管紧张素原基因的多态性，同时用最大期望值方法进行两位点连锁不平衡和三位点的单倍型分析。结果 在M235T和A－20C，M235T和A－6G，A－20C和A－6G位点观察到了连锁不平衡（P＜10^-4)。病例－对照检验显示T235等位基因频率在高血压组中高于对照组，但所有单倍型频率分布在高血压组和正常对照组间差异无显著性。结论 受检人群中AGT基因各单倍型与高血压未见关联，但AGT基因T235位点可能是一种重要的高血压风险因子。     

Identification of a type 1 diabetes-associated CD4 promoter haplotype with high constitutive activity

CD4 is a candidate gene in autoimmune diseases, including Type 1 diabetes mellitus (T1DM), because the CD4 receptor is crucial for appropriate antigen responses of CD4(+) T cells. We previously found linkage between a CD4-1188(TTTTC)(5-14) promoter polymorphism and T1DM. In the present study, we screened the human CD4 promoter for mutations and identified three frequent single nucleotide polymorphisms (SNPs): CD4-181C/G, CD4-521C/G and CD4-1050T/C. The SNPs are in strong linkage disequilibrium (LD) and association with the CD4-1188(TTTTC)(5-14) alleles, and we observed nine CD4 promoter haplotypes, of which four are frequent. We genotyped the SNPs in 253 Danish T1DM families (1129 individuals) and found evidence for linkage and association of a CD4 (A4(-1188)T(-1050)G(-521)C(-181)) haplotype to T1DM. In reporter studies, we show that (1) the T1DM-associated CD4 haplotype encodes high constitutive promoter activity and (2) the CD4-181G variant encodes higher stimulated promoter activity than the CD4-181C variant. This difference is in part neutralized in the frequently occurring CD4 promoter haplotypes by the more upstream genetic variants. Thus, we report functional impact of a novel CD4-181C/G SNP on stimulated CD4 promoter activity and the identification of a novel CD4 haplotype with high constitutive promoter activity that is linked and associated with T1DM.