Context Of Haplotypes Research Articles

A common Alu insertion in the 3'UTR of TMEM106B is associated with risk of dementia.

Sequence variants in TMEM106B have been associated with an increased risk of developing dementia. As part of our efforts to generate a set of mouse lines in which we replaced the mouse Tmem106b gene with a human TMEM106B gene comprised of either a risk or protective haplotype, we conducted an in-depth sequence analysis of these alleles. We also analyzed transcribed TMEM106B sequences using RNA-seq data (AD Knowledge portal) and full genome sequences (1000 Genomes). We identified an AluYb8 insertion in the 3' untranslated region (3'UTR) of the TMEM106B risk haplotype. We found this AluYb8 insertion in every risk haplotype analyzed, but not in either protective haplotypes or in non-human primates. We conclude that this risk haplotype arose early in human development with a single Alu-insertion event within a unique haplotype context. This AluYb8 element may act as a functional variant in conferring an increased risk of developing dementia. We conducted an in-depth sequence analysis of (1) a risk and (2) a protective haplotype of the human TMEM106B gene. We also analyzed transcribed TMEM106B sequences using RNA-seq data (AD Knowledge Portal) and full genome sequences (1000 Genomes). We identified an AluYb8 insertion in the 3' untranslated region (3'UTR) of the TMEM106B risk haplotype. We found this AluYb8 insertion in every risk haplotype analyzed, but not in either protective haplotypes or in non-human primates. This AluYb8 element may act as a functional variant in conferring an increased risk of developing dementia.

Structural analysis of mitochondrial rRNA gene variants identified in patients with deafness.

The last few years have witnessed dramatic advances in our understanding of the structure and function of the mammalian mito-ribosome. At the same time, the first attempts to elucidate the effects of mito-ribosomal fidelity (decoding accuracy) in disease have been made. Hence, the time is right to push an important frontier in our understanding of mitochondrial genetics, that is, the elucidation of the phenotypic effects of mtDNA variants affecting the functioning of the mito-ribosome. Here, we have assessed the structural and functional role of 93 mitochondrial (mt-) rRNA variants thought to be associated with deafness, including those located at non-conserved positions. Our analysis has used the structural description of the human mito-ribosome of the highest quality currently available, together with a new understanding of the phenotypic manifestation of mito-ribosomal-associated variants. Basically, any base change capable of inducing a fidelity phenotype may be considered non-silent. Under this light, out of 92 previously reported mt-rRNA variants thought to be associated with deafness, we found that 49 were potentially non-silent. We also dismissed a large number of reportedly pathogenic mtDNA variants, 41, as polymorphisms. These results drastically update our view on the implication of the primary sequence of mt-rRNA in the etiology of deafness and mitochondrial disease in general. Our data sheds much-needed light on the question of how mt-rRNA variants located at non-conserved positions may lead to mitochondrial disease and, most notably, provide evidence of the effect of haplotype context in the manifestation of some mt-rRNA variants.

Molecular characterization of a large group of Mucopolysaccharidosis type IIIC patients reveals the evolutionary history of the disease.

Mucopolysaccharidosis type IIIC (MPSIIIC) is a severe, rare autosomal recessive disorder caused by variants in the heparan-α-glucosaminide N-acetyltransferase (HGSNAT) gene which result in lysosomal accumulation of heparan sulfate. We analyzed clinical presentation, molecular defects and their haplotype context in 78 (27 novel) MPSIIIC cases from 22 countries, the largest group studied so far. We describe for the first time disease-causing variants in the patients from Brazil, Algeria, Azerbaijan, and Iran, and extend their spectrum within Canada, Colombia, Turkey, and the USA. Six variants are novel: two missense, c.773A>T/p.N258I and c.1267G>T/p.G423W, a nonsense c.164T>A/p.L55*, a splice-site mutation c.494-1G>A/p.[P165_L187delinsQSCYVTQAGVRWHHLGSLQALPPGFTPFSYLSLLSSWNC,P165fs], a deletion c.1348delG/p.(D450fs) and an insertion c.1479dupA/p.(Leu494fs). The missense HGSNAT variants lacked lysosomal targeting, enzymatic activity, and likely the correct folding. The haplotype analysis identified founder mutations, p.N258I, c.525dupT, and p.L55* in the Brazilian state of Paraiba, c.493+1G>A in Eastern Canada/Quebec, p.A489E in the USA, p.R384* in Poland, p.R344C and p.S518F in the Netherlands and suggested that variants c.525dupT, c.372-2G>A, and c.234+1G>A present in cis with c.564-98T>C and c.710C>A rare single-nucleotide polymorphisms, have been introduced by Portuguese settlers in Brazil. Altogether, our results provide insights into the origin, migration roots and founder effects of HGSNAT disease-causing variants, and reveal the evolutionary history of MPSIIIC.

Linked read sequencing resolves complex genomic rearrangements in gastric cancer metastases

BackgroundGenome rearrangements are critical oncogenic driver events in many malignancies. However, the identification and resolution of the structure of cancer genomic rearrangements remain challenging even with whole genome sequencing.MethodsTo identify oncogenic genomic rearrangements and resolve their structure, we analyzed linked read sequencing. This approach relies on a microfluidic droplet technology to produce libraries derived from single, high molecular weight DNA molecules, 50 kb in size or greater. After sequencing, the barcoded sequence reads provide long range genomic information, identify individual high molecular weight DNA molecules, determine the haplotype context of genetic variants that occur across contiguous megabase-length segments of the genome and delineate the structure of complex rearrangements. We applied linked read sequencing of whole genomes to the analysis of a set of synchronous metastatic diffuse gastric cancers that occurred in the same individual.ResultsWhen comparing metastatic sites, our analysis implicated a complex somatic rearrangement that was present in the metastatic tumor. The oncogenic event associated with the identified complex rearrangement resulted in an amplification of the known cancer driver gene FGFR2. With further investigation using these linked read data, the FGFR2 copy number alteration was determined to be a deletion-inversion motif that underwent tandem duplication, with unique breakpoints in each metastasis. Using a three-dimensional organoid tissue model, we functionally validated the metastatic potential of an FGFR2 amplification in gastric cancer.ConclusionsOur study demonstrates that linked read sequencing is useful in characterizing oncogenic rearrangements in cancer metastasis.

Abstract B23: Colombians with high percentage of African ancestry, carrying a specific IL1B haplotype, have increased risk of colorectal cancer

Abstract Background: Colorectal cancer mortality rates are heterogeneous across Colombian territory and this variation could be explained by regional differences in Colombian ethnicity due to our high population admixture. Different frequencies of the interleukin 1 beta (IL1B) genetic variations have been described among diverse ethnical groups, but there are no reports on the possible association of these variants with colorectal cancer (CRC) risk as an effect of the genetic population dynamics in Colombia. Methods: To evaluate the association of IL1B haplotypes with colorectal carcinogenesis, we genotyped 317 CRC cases, 199 adenomatous polyps (AP) and 511 age-sex matched controls recruited from six Colombian cities located either on the coastal or the inner zone of the country. Five IL1B SNPs (-3737, -1464, -511, -31 and +3954) were genotyped and all were in HWE (p>0.05). We used data from previous analysis to estimate individual ancestral genetic fractions using STRUCTURE software based on allele frequencies of 678 autosomal SNPs cross-matched with the HapMap dataset, not in LD, and assuming three distinct population origins (CEU, LWJ and CHB populations) from HapMap project as references. Results: We found a 4-SNPs haplotype block (-3737C/-1464G/-511T/-31C) with high LD associated with CRC risk. Additional analysis revealed that this association persisted after adjusting by confounding variables including ancestry information (OR 1.95; CI 1.12 - 3.38; p =0.02). Interestingly, this association was evident among individuals from the coastal zone of the country and it was related with their higher African ancestry fraction (OR 2.26; 95%CI 1.22-4.18; p =0.01). Our results are in agreement with previous reports showing that this haplotype increase the IL1B promoter activity and that is highly prevalent in African-Americans. Conclusions: To the best of our knowledge, this is the first study seeking for the association between CRC risk and IL1B gene in a haplotype context in a population-based association study in Colombia, an admixed population. Our results represent consistent evidence on how the identification of genetic biomarkers of susceptibility could be used to discriminate populations at increased risk of CRC development. Citation Format: Maria Carolina Sanabria-Salas, Gustavo Hernandez-Suarez, Adriana Umaña-Perez, Martha Lucia Serrano-Lopez, Myriam Sanchez-Gomez, Martha Patricia Rojas, Jovanny Zabaleta. Colombians with high percentage of African ancestry, carrying a specific IL1B haplotype, have increased risk of colorectal cancer. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr B23. doi:10.1158/1538-7755.DISP13-B23

Abstract 1343: Association between IL1B gene haplotypes and colorectal cancer risk in Colombian populations: a case-control study.

Abstract Background: Single nucleotide polymorphisms (SNPs) in cytokines can affect gene expression causing an effect in inflammation and carcinogenesis. There are conflicting results in the association between IL1B gene markers with colorectal cancer (CRC). Most reports on the association of IL1B and CRC have analysed individual SNPs despite several studies have implied that studying the haplotype context is important to understand the biological effect in IL1B gene expression and carcinogenesis. Aim: To evaluate the association of CRC and adenomatous polyps (AP) with IL1B haplotypes in Colombian populations Methods: 308 CRC cases, 208 AP and 524 age-sex matched controls were recruited from six Colombian cities with different CRC risk in a multicenter hospital-based case-control study aiming at identifying the environmental and genetic risk factors for CRC in Colombia. Participants gave written informed consent, donated blood samples and answered guided-questionnaires. Subjects were genotyped for IL1B SNPs: -31, +3954, -3737, -1464 and -511 using TaqMan® SNP Genotyping Assays. Hardy-Weinberg Equilibrium (HWE) was assessed and statistical analysis was done using conditional logistic regression adjusting for risk factors and genetic ancestry. Linkage disequilibrium (LD) was calculated using Haploview 4.0 and D’ and r2 statistics. Recessive and codominant models were explored for haplotype analysis using STATA v11.0. The Ethics Board of The National Cancer Institute of Colombia approved this study Results: Among controls all IL1B SNPs were in HWE (p>0.05). We found one haplotype block including all 4 SNPs in the IL1B gene, except the IL1B+3954 SNP which was left out (MAF=0,14). IL1B-31 and IL1B-511 showed to be in high LD (D’: 0,998; r2: 0,948). Under recessive model, we found an association with an increased risk for PA and CRC with the -31C/-511T/-1464G/-3737C haplotype (OR 2.42 IC 1.21 - 4.84 p<0.01). Stratified analysis of PA and CRC risk separately, showed similar results. This risk decreases when the rare allele of the -1464C was also present in the haplotype (p 0.05). These associations persist after adjusting for confounding Conclusions: The -31C/-511T/-1464G/-3737C haplotype was associated with premalignant and malignant lesions in colon and rectum, interesting the effect of the rare alleles -31C and -511T was countered by the -1464C allele since the haplotype -31C/-511T/-1464C/-3737C showed no association with the disease. These findings are correlated with functional assay studies in which the former haplotype increase promoter activity of the IL1B gene and the later decrease that effect supporting the importance of understanding the haplotype context in association studies. These associations with the disease remained after corrected by Caucasian and African ancestry, meaning that these are true and not due to differences in the genetic background of the Colombian population Citation Format: María Carolina Sanabria-Salas, Gustavo Hernández-Suárez, Adriana Umaña, Martha Lucía Serrano López, Myriam Y. Sánchez, Martha P. Rojas, Jovanny Zabaleta. Association between IL1B gene haplotypes and colorectal cancer risk in Colombian populations: a case-control study. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1343. doi:10.1158/1538-7445.AM2013-1343

HLA and Celiac Disease Susceptibility: New Genetic Factors Bring Open Questions about the HLA Influence and Gene-Dosage Effects

Celiac disease (CD) is a chronic inflammatory disorder triggered after gluten ingestion in genetically susceptible individuals. The major genetic determinants are HLA-DQA1*05 and HLA-DQB1*02, which encode the DQ2 heterodimer. These alleles are commonly inherited in cis with DRB1*03∶01, which is associated with numerous immune-related disorders, in some cases contributing with a different amount of risk depending on the haplotype context. We aimed at investigating those possible differences involving DRB1*03∶01-carrying haplotypes in CD susceptibility. A family (274 trios) and a case-control sample (369 CD cases/461 controls) were analyzed. DRB1*03∶01-carrying individuals were classified according to the haplotype present (ancestral haplotype (AH) 8.1, AH 18.2 or non-conserved haplotype) after genotyping of HLA-DRB1, -DQA1, -DQB1, -B8, TNF -308, TNF -376 and the TNFa and TNFb microsatellites. We observe that the AH 8.1 confers higher risk than the remaining DRB1*03∶01-carrying haplotypes, and this effect only involves individuals possessing a single copy of DQB1*02. CD risk for these individuals is similar to the one conferred by inherit DQA1*05 and DQB1*02 in trans. It seems that an additional CD susceptibility factor is present in the AH 8.1 but not in other DRB1*03∶01-carrying haplotypes. This factor could be shared with individuals possessing DQ2.5 trans, according to the similar risk observed in those two groups of individuals.

ITS and morphology tell different histories about the species of the Sciuro-hypnum reflexum complex (Brachytheciaceae, Bryophyta)

Morphology and ITS relationships are compared for 111 Sciuro-hypnum specimens, 95 of which belong to S. curtum, S. hylotapetum, or the S. reflexum complex. In the latter, S. reflexum is probably ancestral. Within the S. reflexum complex ITS haplotype relationships are congruent with morphology for S. brotheri, S. dovrense, S. glaciale, and S. ornellanum, and support the recognition of the two new species S. sinolatifolium and S. sichuanicum, whereas incongruence occurs for several species. In an ITS haplotype context, S. tromsoeense and S. latifolium are resolved as polyphyletic, and S. altaicum, some specimens of S. oedipodium, and S. curtum–S. starkei intermediates cannot be distinguished from S. reflexum. Sciuro-hypnum fuegianum is molecularly identical and morphologically similar to S. glaciale, and is considered conspecific with the latter. There is no evidence to suggest that ITS a priori is more reliable than morphology, and we therefore suggest that the abundant incongruent patterns reflect either 1) a young age in terms of number of generations in relation to the rate of ITS differentiation and associated incomplete lineage sorting and occasional hybridization events, 2) a faster rate of morphological than ITS evolution, 3) the existence of many lineages that are propagated by selfing, or 4) regional differences in extinction rates during the Pleistocene glaciations. However, additional information is required to decide which of these is/are most important. Finally, S. squarrosum, a morphologically distinct species that molecularly belongs outside the S. reflexum complex, is described as new.

ABCA1 impacts athero-thrombotic risk and 10-year survival in a contemporary secondary prevention setting

ObjectivesWe prospectively investigated the effects of ATP-binding cassette protein-1 (ABCA1) variants on long-term clinical outcome in patients with coronary artery disease (CAD). BackgroundABCA1 is implicated in the etiology of atherothrombosis and may offer a target to reduce cardiovascular risk. However, the impact of ABCA1 on recurrent cardiovascular disease in a secondary prevention setting is as of yet unknown. MethodsWe studied cause-specific 10-year mortality and quantitative coronary angiography data from the Regression GRowth Evaluation Statin Study (REGRESS), comprising 884 male CAD patients genotyped for promoter variants encompassing a proximal regulatory region (rs2422493, rs1800976, rs2740483 and rs1800977). Kaplan–Meier, proportional hazards and haplotype analyses were used to ascertain single-variant and multi-marker effects on absolute risk and extent of CAD. ResultsProtection from 10-year vascular death could be attributed to the rs2422493 genotype (available in 639 patients) T allele with absolute risk decreasing stepwise from 12.2% to 8.6% to 4.7% per each added allele copy, HR 0.64, p=0.03 and HR 0.53, p=0.04 in the TGCC haplotype context. The TGCC (p=0.04) and TCCT (p=0.003) haplotypes exhibited less extensive CAD. ConclusionsOn a background of contemporary secondary prevention, variation in the ABCA1 promoter influences 10-year risk of vascular death and angiographic extent of CAD in men. These insights contribute to identification of patients sharing a specific prognosis, understanding of its etiological basis and development of strategies of risk reduction in CAD.

Identification of a 4.9-kilo base-pair Alu-mediated founder SDHD deletion in two extended paraganglioma families from Austria

Hereditary paraganglioma (PGL) is characterized by the development of highly vascularized paraganglionic tumors as a result of germline mutations in the SDHB, SDHC or SDHD subunit genes of succinate dehydrogenase (SDH; mitochondrial complex II), or in the Von Hippel-Lindau tumor-suppressor gene. Although many PGL mutations have been described, gross SDHD deletions have not yet been implicated as founder mutations and are rarely characterized at the DNA sequence level. We investigated the genetic basis of head and neck PGLs observed in 20 subjects from two unrelated multiplex pedigrees from Austria and identified a 4944-base pair partial SDHD deletion, which escaped PCR-based detection methods. The deletion occurred between Alu elements and was present within the same haplotype context in both pedigrees, indicating a founder effect. The deletion caused tumors only after a paternal transmission similar to other conventional SDHD mutations, suggesting preservation of genomic imprinting mechanisms operating at this locus. These data describe a large SDHD deletion at the genomic sequence level and indicate that gross SDHD deletions could be a founder PGL mutation in certain populations.

Maroteaux–Lamy syndrome: Functional characterization of pathogenic mutations and polymorphisms in the arylsulfatase B gene

Mucopolysaccharidosis VI (MPS VI; Maroteaux-Lamy syndrome) is an autosomal recessive lysosomal disorder caused by deficiency of N-acetylgalactosamine-4-sulfatase (ARSB), which is required for the degradation of dermatan sulfate. We recently reported mutational screening of 12 Spanish and 4 Argentinian MPS VI patients. In the present study, seven missense mutations (c.245T>G [p.L82R], c.413A>G [p.Y138C], c.719C>T [p.S240F], c.922G>A [p.G308R], c.937C>G [p.P313A], c.1340G>T [p.C447F] and c.1415T>C [p.L472P]) were transiently expressed in COS-7 cells and 4-sulfatase activity was measured in cell extracts. All mutations resulted in less than 6% of wild-type enzyme activity, in most cases undetectable. Mutations were expressed in their original haplotype context with respect to two non-synonymous polymorphisms present in the ARSB protein, p.V358M and p.S384N. The three less frequent haplotype combinations yielded an ARSB activity of 16%, 57% and 70%, when compared to the most frequent haplotype (p.358V and p.384S). Western blot analyses showed that the expressed mutations significantly reduced the amount of mature protein. Sub-cellular localization studies of mutant ARSB proteins in fibroblasts of MPS VI patients were performed. RNA analysis confirmed that nonsense-mediated RNA decay had taken place for all mutant alleles (c.1143−1G>C, c.1143−8T>G, p.W322X, c.427delG and c.1142+2T>A) which were candidates for causing RNA degradation by this mechanism. In summary, all the ARSB mutations studied had a significant effect on enzyme activity, protein processing and/or mRNA stability.

Functional Haplotype Frequencies of the Interleukin-1B Promoter in the Korean Population

Abstract Single nucleotide polymorphisms (SNPs) in the promoter region of the IL-1B (interleukin-1) gene have been im-plicated in a variety of diseases that have an in-flammatory component. However, there has been sig-nificant heterogeneity among study results, especially between Caucasian and Asian populations. Recently, it has been reported that SNPs in the IL-1B gene affect transcription, according to haplotype context, and ge-netic association studies may be more informative if functional SNP haplotypes of population are analyzed. Therefore, we estimated the distribution of IL-1B pro-moter haplotypes in 433 Koreans using the three major functional IL-1B promoter SNPs (IL-1B-1464, -511, and -31) and compared the results with those in Caucasians. The difference in IL-1B promoter haplotype frequency between Korean and Caucasian populations was stat-istically significant. The potentially more inflammatory haplotypes had higher frequencies in Koreans when compared with Caucasians. These Korean haplotype da-ta will be useful for future association studies between IL-1B SNPs and disease risk.Keywords: Interleukin-1B; polymorphism; haplotype; Ko-reanSNPs in the promoter region of IL-1B gene have been implicated in a variety of diseases that have an in-flammatory component, including cardiovascular dis-ease, gastric cancer, Alzheimer disease, and periodontal disease (Iacoviello et al., 2005; Camargo et al., 2006; Griffin et al., 2000; Kornman et al., 1997). The IL-1B gene is highly polymorphic, and several SNPs have been frequently associated with several diseases (Iacoviello et al., 2005; Francis et al, 1999; El-Omar et al., 2000). However, there has been significant hetero-geneity among study results, especially between Caucasian and Asian populations (Camargo et al., 2006; Lee et al., 2004; Yang et al., 2004; Zeng et al., 2003; Chang et al., 2005; Ma et al., 2003; Hodge et al., 2001). Recently, Chen et al (Chen et al., 2006) reported that SNPs in the IL-1B gene affect transcription according to haplotype context, and genetic association studies may be more informative if functional SNP haplotypes are analyzed, rather than individual functional SNPs. Further, they showed that the functional haplotypes differ by ethnic population. These findings underlie the relevance of population haplotypes in the design of genetic stud-ies (Chen et al., 2006). In view of these findings, we es-timated the distribution of IL-1B promoter haplotypes in 433 Koreans using three major functional IL-1B pro-moter SNPs (IL-1B-1464, -511, and -31) and compared the results with those in Caucasian and African populations. Although the IL-1B-3737 polymorphism al-so had allele-specific differences in nuclear protein bind-ing, no differences in promoter activity were observed with different alleles of this SNP on a background of the other SNP sets that showed higher promoter activity (Chen et al., 2006). Because it has been suggested that the functional significance of IL-1B-3737 might depend on a broader haplotype, we used the three SNPs for haplotype analysis. Haplotypes were reconstructed by PHASE version 2.1, using previously produced genotype data (Lee et al., 2004). Of the possible eight haplotypes, three common ones accounted for ≥98% of the estimated haplotypes in the Korean population. Table 1 shows the haplotype fre-quency estimation in each population. The potentially more inflammatory IL-1B-511T/-31C haplotype repre-sented 53.5% of the Korean haplotypes, compared with 33.7% of the Caucasian haplotypes. So far, in many previous association studies, the individual SNP ap-proach, most frequently using IL-1B-511 and IL-1B-31, has been adopted. To our knowledge, we reported first that the IL-1B-1464 polymorphism has allele-specific dif-ferences in nuclear protein binding and is associated with a clinical disease (Lee et al., 2004). The biological implication of this polymorphism was supported by in vivo studies by Chen et al. that showed that the IL-1B-1464 polymorphism has substantial allele-specific effects when both IL-1B-511 and IL-1B-31 were alleles T and C, respectively (Chen et al., 2006). The more in-formative haplotype 1 (GTC), containing the IL-1B-1464 polymorphism, which shows the highest transcriptional activity, represents 9.3% and 6.0% of Korean and Caucasian haplotypes, respectively, whereas haplotype

IL1B gene promoter haplotype pairs predict clinical levels of interleukin-1β and C-reactive protein

Interleukin-1beta (IL-1beta) activates inflammatory mediator cascades and has been implicated in the pathogenesis of several diseases. Single nucleotide polymorphisms (SNPs) of the IL1B promoter have been associated with various inflammatory diseases. We recently reported that IL1B gene transcription was influenced by four promoter SNPs, and that individual SNP function in vitro was governed by haplotype context. In the present study we tested the in vivo relevance of this observation by comparing IL1B promoter haplotype-pairs with IL-1beta protein levels in 900 gingival tissue fluid samples. Three SNPs (-511, -1464, -3737) defined four IL1B promoter haplotypes that occurred in the study population and could be assigned unambiguously to each chromosome. The four haplotypes defined ten haplotype-pairs of which four pairs, representing 57% of the population, were associated with 28-52% higher IL-1beta protein levels in vivo. Two of these pairs, characterized by homozygosity for the common allele at -3737, were also associated with raised serum levels of C-reactive protein (p = 0.02). We validated these findings in stimulated peripheral blood mononuclear cells (PBMCs) from a separate population (N = 70). PBMCs with IL1B haplotype-pairs associated with higher in vivo levels of IL-1beta produced 86-287% more IL-1beta in vitro than the reference group. We believe that this is the first demonstration of a relationship between in vivo levels of an inflammatory mediator and gene promoter haplotypes on both chromosomes. These findings may apply to other inducible genes and could provide a logical framework for exploring disease risk related to genetic variability in pathogenic mediators.

Genetic Variation Within the β1-Adrenergic Receptor Gene Results in Haplotype-Specific Expression Phenotypes

Cardiac beta1-adrenergic receptor (beta1AR) responsiveness in heart failure exhibits interindividual variation that may be attributable to polymorphisms of the intronless beta1AR gene. We sought to ascertain the polymorphisms of the full-length gene and the specific combinations of polymorphisms (haplotypes) in two reference populations. Using whole-gene transfections, we established the impact of beta1AR polymorphisms, within the context of haplotypes, on receptor expression. Fifteen polymorphisms within the 6.1-kb gene with allele frequencies > or =0.05 were found in the 5'-flanking and coding regions, but none in the 3'UTR. These were organized into six common haplotypes. Ethnic-specific and cosmopolitan polymorphisms and haplotypes were noted. Whole-gene transfections of A431 cells revealed an association between haplotype and expression, with as much as twofold differences in expression. Phenotypes clustered into three groups, representing high (two haplotypes), intermediate (three haplotypes), and low (one haplotype) expression. We conclude that the beta1AR gene is highly polymorphic and is commonly found in six haplotypic forms in the population. Receptor expression varies by haplotype, which provides the foundation for cardiovascular association studies with enhanced predictive power using beta1AR haplotypes, or haplotype expression clusters, as compared with individual polymorphisms.

Single nucleotide polymorphisms in the proximal promoter region of apolipoprotein M gene (apoM) confer the susceptibility to development of type 2 diabetes in Han Chinese

Dyslipidemia correlating to insulin resistance is one of the key features in type 2 diabetes (T2D). Recent studies have demonstrated that apolipoprotein M (apoM) is important for the formation of prebeta-high-density lipoprotein (HDL) and cholesterol (CHO) efflux in macrophages to HDL. In the present study, we investigated the potential association of apoM genetic variation with the development of T2D. Single nucleotide polymorphisms (SNPs) C-1065A, T-855C and T-778C in the proximal promoter region of apoM gene were validated to represent in Han Chinese. Further genotyping experiments in 170 T2D patients and 156 non-diabetic control subjects were performed with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Single marker analysis for SNP T-778C indicated that T2D patients had increased frequency of C allele in comparison with non-diabetic controls (10.6% versus 5.8%, P = 0.026, OR = 1.934). In non-diabetic controls, the carriers with CT and CC genotypes had higher plasma CHO (221.7 versus 204.2 mg/dL, P = 0.033) and fasting plasma glucose (FPG) (92.6 versus 89.7 mg/dL, P = 0.041) levels than the subjects with TT genotype. Further analysis with adjustment for age, BMI, SBP, DBP, CHO and TG demonstrated that this SNP was strongly associated with T2D (P = 0.013, OR = 2.287). Haplotype analysis for those three SNPs, however, indicated that the common haplotypes were less informative than studying the role of the T-778C variant independently of the haplotype context. The present study provided the first evidence that SNP T-778C in the proximal promoter region of apoM gene was associated with the levels of plasma CHO and FPG and also conferred the risk in the development of T2D among Han Chinese.

Single nucleotide polymorphisms in the human interleukin-1B gene affect transcription according to haplotype context

We questioned the significance of haplotype structure in gene regulation by testing whether individual single nucleotide polymorphisms (SNPs) within a gene promoter region [interleukin-1-beta (IL1B)] might affect promoter function and, if so, whether function was dependent on haplotype context. We sequenced genomic DNA from 25 individuals of diverse ethnicity, focusing on exons and upstream flanking regions of genes of the cluster. We identified four IL1B promoter region SNPs that were active in transient transfection reporter gene assays. To substantiate allelic differences found in reporter gene assays, we also examined nuclear protein binding to promoter sequence oligonucleotides containing different alleles of the SNPs. The effect of individual SNPs on reporter gene transcription varied according to which alleles of the three other SNPs were present in the promoter construct. The SNP patterns that influenced function reflected common haplotypes that occur in the population, suggesting functionally significant interactions between SNPs according to haplotype context. Of the haplotypes that include the four functional IL1B promoter SNPs (-3737, -1464, -511, -31), the four haplotypes that showed different contextual effects on SNP function accounted for >98% of the estimated haplotypes in Caucasian and African-American populations. This finding underlines the importance of understanding the haplotype structure of populations used for genetic studies and may be especially important in the functional analysis of genetic variation across gene regulatory regions.

Evaluating the informative power of Y-STRs: a comparative study using European and new African haplotype data

Male individuals from Maputo (Mozambique) were sampled and 18 Y-STRs were typed: the nine currently used to define the “minimal haplotype” employed in the European, American and Asian “Y-STR Haplotype Reference Databases”, as well as the recently described DYS434, DYS437, DYS438, DYS439, DYS460, DYS461, GATA A10, GATA C4 and GATA H4. Allele and haplotype frequencies were estimated in a sample of 112 individuals, where it was possible to define 101 haplotypes, with an observed haplotype diversity (HD) of 0.9973. Allele diversity varied between 0.0179 and 0.9220, DYS385 showing the highest level of polymorphism and DYS392 the lowest. When considering only the most recent Y-STRs, the degree of diversity varied between 0.4011 (DYS438) and 0.6910 (GATA C4), except for DYS434 and DYS437 where a very low diversity was observed (0.0700 and 0.0526, respectively). When analysing the same 112 individuals for the nine Y-STRs included in the minimal haplotype, 78 haplotypes were distinguished with a corresponding observed diversity of 0.9884, a considerably lower value than those for Northern Portugal ( n=208; HD: 0.9925) and Macao ( n=63; HD: 0.9990). Concerning all 18 Y-STRs studied in this population, the observed diversity demonstrates their usefulness in forensic applications, with the exception of DYS434, DYS437 and DYS392. However, since the informative power of a marker has to be judged in haplotype context, a simple software, allowing the evaluation of the increase of HD through the addition of any combination of new markers to the minimum haplotype was designed. The statistical approach devised, demonstrates that an increment on HD is more rapidly obtained for the Mozambican database when adding GATA A10 or DYS439, DYS460, GATA C4, DYS461 or GATA H4, in this order, to the minimal haplotype. DYS434, DYS437 and DYS438, in conjunction with all the other 15 Y-STRs, do not contribute to an increment on HD. When applying the same approach to an European sample (Northern Portugal), the first three Y-STR choices coincide, but the next order of markers are GATA H4, then DYS437 and finally DYS461. In this sample, DYS434, DYS438 and GATA C4 do not increment HD any further.