Kelly A. Volcik

Germ-Line Mutational Analysis of the TSC2 Gene in 90 Tuberous-Sclerosis Patients

Ninety patients with tuberous-sclerosis complex (TSC) were tested for subtle mutations in the TSC2 gene, by means of single-strand conformational analysis (SSCA) of genomic DNA. Patients included 56 sporadic cases and 34 familial probands. For all patients, SSCA was performed for each of the 41 exons of the TSC2 gene. We identified 32 SSCA changes, 22 disease-causing mutations, and 10 polymorphic variants. Interestingly, we detected mutations at a much higher frequency in the sporadic cases (32%) than in the multiplex families (9%). Among the eight families for which linkage to the TSC2 region had been determined, only one mutation was found. Mutations were distributed equally across the gene; they included 5 deletions, 3 insertions, 10 missense mutations, 2 nonsense mutations, and 2 tandem duplications. We did not detect an increase in mutations either in the GTPase-activating protein (GAP)-related domains of TSC2 or in the activating domains that have been identified in rat tuberin. We did not detect any mutations in the exons (25 and 31) that are spliced out in the isoforms. There was no evidence for correspondence between variability of phenotype and type of mutation (missense versus early termination). Diagnostic testing will be difficult because of the genetic heterogeneity of TSC (which has at least two causative genes: TSC1 and TSC2), the large size of the TSC2 gene, and the variety of mutations. More than half of the mutations that we identified (missense, small in-frame deletion, and tandem duplication) are not amenable to the mutation-detection methods, such as protein-truncation testing, that are commonly employed for genes that encode proteins with tumor-suppressor function.

Association of novel genetic loci with circulating fibrinogen levels a genome-wide association study in 6 population-based cohorts

Background— Fibrinogen is both central to blood coagulation and an acute-phase reactant. We aimed to identify common variants influencing circulation fibrinogen levels. Methods and Results— We conducted a genome-wide association analysis on 6 population-based studies, the Rotterdam Study, the Framingham Heart Study, the Cardiovascular Health Study, the Atherosclerosis Risk in Communities Study, the Monitoring of Trends and Determinants in Cardiovascular Disease/KORA Augsburg Study, and the British 1958 Birth Cohort Study, including 22 096 participants of European ancestry. Four loci were marked by 1 or more single-nucleotide polymorphisms that demonstrated genome-wide significance ( P <5.0×10−8). These included a single-nucleotide polymorphism located in the fibrinogen β chain ( FGB ) gene and 3 single-nucleotide polymorphisms representing newly identified loci. The high-signal single-nucleotide polymorphisms were rs1800789 in exon 7 of FGB ( P =1.8×10−30), rs2522056 downstream from the interferon regulatory factor 1 ( IRF1 ) gene ( P =1.3×10−15), rs511154 within intron 1 of the propionyl coenzyme A carboxylase ( PCCB ) gene ( P =5.9×10−10), and rs1539019 on the NLR family pyrin domain containing 3 isoforms ( NLRP3 ) gene ( P =1.04×10−8). Conclusions— Our findings highlight biological pathways that may be important in regulation of inflammation underlying cardiovascular disease. Received October 1, 2008; accepted January 5, 2009. # CLINICAL PERSPECTIVE {#article-title-2}Background—Fibrinogen is both central to blood coagulation and an acute-phase reactant. We aimed to identify common variants influencing circulation fibrinogen levels. Methods and Results—We conducted a genome-wide association analysis on 6 population-based studies, the Rotterdam Study, the Framingham Heart Study, the Cardiovascular Health Study, the Atherosclerosis Risk in Communities Study, the Monitoring of Trends and Determinants in Cardiovascular Disease/KORA Augsburg Study, and the British 1958 Birth Cohort Study, including 22 096 participants of European ancestry. Four loci were marked by 1 or more single-nucleotide polymorphisms that demonstrated genome-wide significance (P<5.0×10−8). These included a single-nucleotide polymorphism located in the fibrinogen &bgr; chain (FGB) gene and 3 single-nucleotide polymorphisms representing newly identified loci. The high-signal single-nucleotide polymorphisms were rs1800789 in exon 7 of FGB (P=1.8×10−30), rs2522056 downstream from the interferon regulatory factor 1 (IRF1) gene (P=1.3×10−15), rs511154 within intron 1 of the propionyl coenzyme A carboxylase (PCCB) gene (P=5.9×10−10), and rs1539019 on the NLR family pyrin domain containing 3 isoforms (NLRP3) gene (P=1.04×10−8). Conclusions—Our findings highlight biological pathways that may be important in regulation of inflammation underlying cardiovascular disease.

Diabetes Genes and Prostate Cancer in the Atherosclerosis Risk in Communities Study

There is a known inverse association between type 2 diabetes (T2D) and prostate cancer (PrCa) that is poorly understood. Genetic studies of the T2D-PrCa association may provide insight into the underlying mechanisms of this association. We evaluated associations in the Atherosclerosis Risk in Communities study between PrCa and nine T2D single nucleotide polymorphisms from genome-wide association studies of T2D (in CDKAL1, CDKN2A/B, FTO, HHEX, IGF2BP2, KCNJ11, PPARG, SLC30A8, and TCF7L2) and four T2D single nucleotide polymorphisms from pre–genome-wide association studies (in ADRB2, CAPN10, SLC2A2, and UCP2). From 1987 to 2000, there were 397 incident PrCa cases among 6,642 men ages 45 to 64 years at baseline. We used race-adjusted Cox proportional hazards models to estimate associations between PrCa and increasing number of T2D risk-raising alleles. PrCa was positively associated with the CAPN10 rs3792267 G allele [hazard ratio (HR) 1.20; 95% confidence interval (CI), 1.00-1.44] and inversely associated with the SLC2A2 rs5400 Thr110 allele (HR, 0.85; 95% CI, 0.72, 1.00), the UCP2 rs660339 Val55 allele (HR, 0.84; 95% CI, 0.73, 0.97) and the IGF2BP2 rs4402960 T allele (HR, 0.79; 95% CI, 0.61-1.02; blacks only). The TCF7L2 rs7903146 T allele was inversely associated with PrCa using a dominant genetic model (HR, 0.79; 95% CI, 0.65-0.97). Further knowledge of T2D gene-PrCa mechanisms may improve understanding of PrCa etiology. Cancer Epidemiol Biomarkers Prev; 19(2); 558–65

Genetic Variants Identified in a European Genome-Wide Association Study That Were Found to Predict Incident Coronary Heart Disease in the Atherosclerosis Risk in Communities Study

In 2007, the Wellcome Trust Case Control Consortium (WTCCC) performed a genome-wide association study in 2,000 British coronary heart disease (CHD) cases and 3,000 controls after genotyping 469,557 single nucleotide polymorphisms (SNPs). Seven variants associated with CHD were initially identified, and 5 SNPs were later found in replication studies. In the current study, the authors aimed to determine whether the 12 SNPs reported by the WTCCC predicted incident CHD through 2004 in a biracial, prospective cohort study (Atherosclerosis Risk in Communities) comprising 15,792 persons aged 45-64 years who had been selected by probability sampling from 4 different US communities in 1987-1989. Cox proportional hazards models with adjustment for age and gender were used to estimate CHD hazard rate ratios (HRRs) over a 17-year period (1,362 cases in whites and 397 cases in African Americans) under an additive genetic model. The results showed that 3 SNPs in whites (rs599839, rs1333049, and rs501120; HRRs were 1.10 (P = 0.044), 1.14 (P < 0.001), and 1.14 (P = 0.030), respectively) and 1 SNP in African Americans (rs7250581; HRR = 1.60, P = 0.05) were significantly associated with incident CHD. This study demonstrates that genetic variants revealed in a case-control genome-wide association study enriched for early disease onset may play a role in the genetic etiology of CHD in the general population.

Examinations of Methylenetetrahydrofolate Reductase C677T and A1298C Mutations—and In Utero Viability

To the Editor:The recently published study by Isotalo et al. (2000xNeonatal and fetal methylenetetrahydrofolate reductase genetic polymorphisms: an examination of C677T and A1298C mutations. Isotalo, PA, Wells, GA, and Donnelly, JG. Am J Hum Genet. 2000; 67: 986–990Abstract | Full Text | Full Text PDF | PubMed | Scopus (162)See all References2000) analyzed the methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C mutations in neonatal and fetal groups, to determine whether particular MTHFR genotype combinations are associated with decreased in utero viability. Isotalo et al. (2000xNeonatal and fetal methylenetetrahydrofolate reductase genetic polymorphisms: an examination of C677T and A1298C mutations. Isotalo, PA, Wells, GA, and Donnelly, JG. Am J Hum Genet. 2000; 67: 986–990Abstract | Full Text | Full Text PDF | PubMed | Scopus (162)See all References2000) observed all possible genotype combinations in the fetal group, but combined 677CT/1298CC and 677TT/1298CC genotypes were not observed in the neonatal group. Therefore, they hypothesized that decreased viability exists among fetuses carrying the 677CT/1298CC and 677TT/1298CC genotypes, with a possible selection disadvantage in fetuses with an increased number of mutant MTHFR alleles. They also did not observe the 677CT/1298CC and 677TT/1298CC genotypes in a population consisting of healthy adult controls.We have tested for the MTHFR C677T and A1298C mutations in a Hispanic population of Mexican descent, to determine risk for spina bifida (SB) (Volcik et al. 2000xMethylenetetrahydrofolate reductase and spina bifida: an evaluation of level of defect and maternal genotype risk in Hispanics. Volcik, KA, Blanton, SH, Tyerman, GH, Jong, ST, Rott, EJ, Page, TZ, Romaine, NK, and Northrup, H. Am J Med Genet. 2000; 95: 21–27Crossref | PubMed | Scopus (57)See all References2000). Although we observed all possible MTHFR 677/1298 genotype combinations in this Hispanic population, the 677TT/1298CC combination was observed only once, in the mother of an affected individual (table 1table 1). We have analyzed the MTHFR C677T and A1298C mutations in a U.S. population of European descent, composed of patients with SB, their parents, and controls, and have observed the 677CT/1298CC genotype combination (table 2table 2). In addition, we have observed the MTHFR C677T and A1298C mutations in a Canadian population of European descent, composed of patients with SB and their parents (table 3table 3). We observed only a single individual, a patient with SB, with the 677TT/1298AC genotype. However, because of the small size of our sample, we expected that only one or two individuals in each of the groups would have the 677TT/1298AC genotype. It is therefore difficult to reach conclusions, on the basis of the absence of this genotype in the small Canadian population that we studied. The presence of these genotypes in healthy parents and controls militates against the hypothesis, proposed by Isotalo et al. (2000xNeonatal and fetal methylenetetrahydrofolate reductase genetic polymorphisms: an examination of C677T and A1298C mutations. Isotalo, PA, Wells, GA, and Donnelly, JG. Am J Hum Genet. 2000; 67: 986–990Abstract | Full Text | Full Text PDF | PubMed | Scopus (162)See all References2000), that the absence of the 677CT/1298CC genotype suggests that “additional MTHFR mutations in cis are potentially deleterious or lethal” (Isotalo et al. 2000xNeonatal and fetal methylenetetrahydrofolate reductase genetic polymorphisms: an examination of C677T and A1298C mutations. Isotalo, PA, Wells, GA, and Donnelly, JG. Am J Hum Genet. 2000; 67: 986–990Abstract | Full Text | Full Text PDF | PubMed | Scopus (162)See all References2000, p. 989). Perhaps it is the combination of two mutant alleles at both sites (677TT/1298CC), reaching a threshold of four, rather than three, mutations that creates a disadvantage. Other groups have also identified individuals with the 677T and 1298C alleles in the cis configuration and individuals with the mutations in the trans configuration (Weisberg et al. 1998xA second genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity. Weisberg, I, Tran, P, Christensen, B, Sibani, S, and Rozen, R. Mol Genet Metab. 1998; 64: 169–172Crossref | PubMed | Scopus (888)See all References1998; Friedman et al. 1999xA common mutation A1298C in human methylenetetrahydrofolate reductase gene: association with plasma total homocysteine and folate concentrations. Friedman, G, Goldschmidt, N, Friedlander, Y, Ben-Yehuda, A, Selhub, J, Babaey, S, Mendel, M, Kidron, M, and Bar-On, H. J Nutr. 1999; 129: 1656–1661PubMedSee all References1999).Table 1Combined MTHFR C677T/A1298C Genotype or Allele Frequencies in a Hispanic Population of Mexican Descent, Composed of Patients with SB, Their Parents, and ControlsObserved Frequency inGenotype or AllelePatients (n = 302)Mothers (n = 281)Fathers (n = 143)Controls (n = 82)MTHFR C677T/A1298C genotype: CT/CC.003.000.007.012 TT/AC.020.028.021.024 TT/CC.000.004.000.000MTHFR allele: 677C.493.477.549.527 677T.507.523.451.473 1298A.854.856.815.811 1298C.146.144.185.189Table 2Combined MTHFR C677T/A1298C Genotype or Allele Frequencies in a U.S. Population of European Descent, Composed of Patients with SB, Their Parents, and ControlsObserved Frequency inGenotype or AllelePatients (n = 160)Mothers (n = 149)Fathers (n = 107)Controls (n = 66)MTHFR C677T/A1298C genotype: CT/CC.000.020.019.015 TT/AC.038.007.019.000 TT/CC.000.000.000.000MTHFR allele: 677C.578.601.551.682 677T.422.409.449.318 1298A.694.721.734.712 1298C.306.279.266.288Table 3Combined MTHFR C677T/A1298C Genotype or Allele Frequencies in a Canadian Population of European Descent, Composed of Patients with SB and Their ParentsObserved Frequency inGenotype or AllelePatients (n = 46)Mothers (n = 45)Fathers (n = 30)MTHFR C677T/A1298C genotype: CT/CC.022.067.033 TT/AC.022.000.000 TT/CC.000.000.000MTHFR allele: 677C.641.722.683 677T.359.278.317 1298A.696.589.667 1298C.304.411.333An additional concern is that Isotalo et al. (2000xNeonatal and fetal methylenetetrahydrofolate reductase genetic polymorphisms: an examination of C677T and A1298C mutations. Isotalo, PA, Wells, GA, and Donnelly, JG. Am J Hum Genet. 2000; 67: 986–990Abstract | Full Text | Full Text PDF | PubMed | Scopus (162)See all References2000) fail to indicate the ethnicity of the population that they studied. Therefore, we have provided data from three ethnic groups (Hispanics of Mexican descent, U.S. individuals of European descent, and Canadians of European descent), to compare genotype and allele frequencies. If the population studied by Isotalo et al. (2000xNeonatal and fetal methylenetetrahydrofolate reductase genetic polymorphisms: an examination of C677T and A1298C mutations. Isotalo, PA, Wells, GA, and Donnelly, JG. Am J Hum Genet. 2000; 67: 986–990Abstract | Full Text | Full Text PDF | PubMed | Scopus (162)See all References2000) was Canadian, it is notable that they did not observe the 677CT/1298CC genotype in their neonatal group, whereas we observed this genotype in 2%–6% of the Canadian population, of patients with SB and their parents, that we studied. Our data support the conclusion of Isotalo et al. (2000xNeonatal and fetal methylenetetrahydrofolate reductase genetic polymorphisms: an examination of C677T and A1298C mutations. Isotalo, PA, Wells, GA, and Donnelly, JG. Am J Hum Genet. 2000; 67: 986–990Abstract | Full Text | Full Text PDF | PubMed | Scopus (162)See all References2000) concerning decreased viability among fetuses with the 677TT/1298CC genotype, because we did not observe this genotype in the U.S. and Canadian populations that we studied. Because we observed, in three different populations, the 677CT/1298CC genotype at frequencies nearing those expected, we conclude that this genotype does not result in a significant selective disadvantage.

Deletion of ADORA2B from myeloid cells dampens lung fibrosis and pulmonary hypertension

Idiopathic pulmonary fibrosis (IPF) is a lethal, fibroproliferative disease. Pulmonary hypertension (PH) can develop secondary to IPF and increase mortality. Alternatively, activated macrophages (AAMs) contribute to the pathogenesis of both IPF and PH. Here we hypothesized that adenosine signaling through the ADORA2B on AAMs impacts the progression of these disorders and that conditional deletion of ADORA2B on myeloid cells would have a beneficial effect in a model of these diseases. Conditional knockout mice lacking ADORA2B on myeloid cells (Adora2Bf/f‐LysMCre) were exposed to the fibrotic agent bleomycin (BLM; 0.035 U/g body weight, i.p.). At 14, 17, 21, 25, or 33 d after exposure, SpO2, bronchoalveolar lavage fluid (BALF), and histologic analyses were performed. On day 33, lung function and cardiovascular analyses were determined. Markers for AAM and mediators of fibrosis and PH were assessed. Adora2Bf/f‐LysMCre mice presented with attenuated fibrosis, improved lung function, and no evidence of PH compared with control mice exposed to BLM. These findings were accompanied by reduced expression of CD206 and arginase‐1, markers for AAMs. A 10‐fold reduction in IL‐6 and a 5‐fold decrease in hyaluronan, both linked to lung fibrosis and PH, were also observed. These data suggest that activation of the ADORA2B on macrophages plays an active role in the pathogenesis of lung fibrosis and PH.‐Karmouty‐Quintana, H., Philip, K., Acero, L. F., Chen, N.‐Y., Weng, T., Molina, J. G., Luo, F., Davies, J., Le, N.‐B., Bunge, I., Volcik, K. A., Le, T.‐T. T., Johnston, R. A., Xia, Y., Eltzschig, H. K., Blackburn, M. R. Deletion of ADORA2B from myeloid cells dampens lung fibrosis and pulmonary hypertension. FASEB J. 29, 50–60 (2015). www.fasebj.org

Gene-Centric Meta-Analysis of Lipid Traits in African, East Asian and Hispanic Populations

Meta-analyses of European populations has successfully identified genetic variants in over 100 loci associated with lipid levels, but our knowledge in other ethnicities remains limited. To address this, we performed dense genotyping of ∼2,000 candidate genes in 7,657 African Americans, 1,315 Hispanics and 841 East Asians, using the IBC array, a custom ∼50,000 SNP genotyping array. Meta-analyses confirmed 16 lipid loci previously established in European populations at genome-wide significance level, and found multiple independent association signals within these lipid loci. Initial discovery and in silico follow-up in 7,000 additional African American samples, confirmed two novel loci: rs5030359 within ICAM1 is associated with total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) (p = 8.8×10−7 and p = 1.5×10−6 respectively) and a nonsense mutation rs3211938 within CD36 is associated with high-density lipoprotein cholesterol (HDL-C) levels (p = 13.5×10−12). The rs3211938-G allele, which is nearly absent in European and Asian populations, has been previously found to be associated with CD36 deficiency and shows a signature of selection in Africans and African Americans. Finally, we have evaluated the effect of SNPs established in European populations on lipid levels in multi-ethnic populations and show that most known lipid association signals span across ethnicities. However, differences between populations, especially differences in allele frequency, can be leveraged to identify novel signals, as shown by the discovery of ICAM1 and CD36 in the current report.

Association of the Complement Factor H Y402H Polymorphism With Cardiovascular Disease Is Dependent Upon Hypertension Status: The ARIC Study

BACKGROUND Complement factor H (CFH) is a plasma protein that is essential in the regulation of the alternative complement pathway and has been implicated as taking part in complement inhibition in atherogenesis. We evaluated the association of the Y402H polymorphism with incident coronary heart disease (CHD), incident ischemic stroke, and carotid artery wall thickness (intima-media thickness (IMT)) in the Atherosclerosis Risk in Communities (ARIC) cohort. METHODS Incident ischemic stroke and CHD were identified through annual telephone calls and hospital and death certificate surveillance. Carotid IMT was measured by means of high-resolution B-mode ultrasound. Four hundred eighty-three validated ischemic stroke and 1,544 CHD events were identified. Because of allele frequency differences between whites and African Americans, analyses were performed separately according to the racial group. RESULTS The 402HH homozygous genotype was a significant predictor of incident ischemic stroke in whites (hazard rate ratio (HRR) 1.47, 95% confidence interval (CI) 1.05-2.05). Significant interaction effects between genotype and hypertension were observed for CHD in whites and for cIMT in whites and African Americans. In further analyses of incident CHD, genotypes carrying the 402H allele were a significant predictor of incident CHD in whites who had hypertension (402YH: HRR 1.19, 95% CI 1.01-1.40; 402HH: HRR 1.28, 95% CI 1.04-1.57). The 402H allele was also associated with higher cIMT measures for whites in the overall cohort, and for whites with hypertension. CONCLUSION The CFH 402H allele was associated with an increased risk for incident CHD and ischemic stroke in whites, with the strength and significance of the association dependent upon hypertension status.

Failure to replicate an association of SNPs in the oxidized LDL receptor gene (OLR1) with CAD

BackgroundThe lectin-like oxidized LDL receptor LOX-1 (encoded by OLR1) is believed to play a key role in atherogenesis and some reports suggest an association of OLR1 polymorphisms with myocardial infarction (MI). We tested whether single nucleotide polymorphisms (SNPs) in OLR1 are associated with clinically significant CAD in the Atherosclerotic Disease, VAscular FuNction, & Geneti C Epidemiology (ADVANCE) study.MethodsADVANCE is a population-based case-control study of subjects receiving care within Kaiser Permanente of Northern California including a subset of participants of the Coronary Artery Risk Development in Young Adults (CARDIA) study. We first resequenced the promoter, exonic, and splice site regions of OLR1 and then genotyped four single nucleotide polymorphisms (SNPs), including a non-synonymous SNP (rs11053646, Lys167Asn) as well as an intronic SNP (rs3736232) previously associated with CAD.ResultsIn 1,809 cases with clinical CAD and 1,734 controls, the minor allele of the coding SNP was nominally associated with a lower odds ratio (OR) of CAD across all ethnic groups studied (minimally adjusted OR 0.8, P = 0.007; fully adjusted OR 0.8, P = 0.01). The intronic SNP was nominally associated with an increased risk of CAD (minimally adjusted OR 1.12, p = 0.03; fully adjusted OR 1.13, P = 0.03). However, these associations were not replicated in over 13,200 individuals (including 1,470 cases) in the Atherosclerosis Risk in Communities (ARIC) study.ConclusionOur results do not support the presence of an association between selected common SNPs in OLR1 and the risk of clinical CAD.

Multi-Ethnic Analysis of Lipid-Associated Loci: The NHLBI CARe Project

Background Whereas it is well established that plasma lipid levels have substantial heritability within populations, it remains unclear how many of the genetic determinants reported in previous studies (largely performed in European American cohorts) are relevant in different ethnicities. Methodology/Principal Findings We tested a set of ∼50,000 polymorphisms from ∼2,000 candidate genes and genetic loci from genome-wide association studies (GWAS) for association with low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) in 25,000 European Americans and 9,000 African Americans in the National Heart, Lung, and Blood Institute (NHLBI) Candidate Gene Association Resource (CARe). We replicated associations for a number of genes in one or both ethnicities and identified a novel lipid-associated variant in a locus harboring ICAM1. We compared the architecture of genetic loci associated with lipids in both African Americans and European Americans and found that the same genes were relevant across ethnic groups but the specific associated variants at each gene often differed. Conclusions/Significance We identify or provide further evidence for a number of genetic determinants of plasma lipid levels through population association studies. In many loci the determinants appear to differ substantially between African Americans and European Americans.