To determine if differences in topographic progression between unaffected keratoconus relatives and normal controls can predict factors associated with the development of keratoconus in a longitudinal study.
We recruited 369 unaffected keratoconus relatives and 119 normal controls in Los Angeles. Both eyes of subjects were examined at baseline clinically and by quantitative videokeratography and at a period ranging from 1 year to 8 years. Progression to keratoconus was evaluated by quantitative videokeratography variables.
Unaffected relatives had higher Central K (CK), I-S and KISA values and were younger than normal controls (CK: 44.70 vs 44.01, P<0.01; I-S: 0.76 vs 0.58, P<0.01, KISA: 29.97 vs 23.89, P=0.02; age: 34.8 vs 41.0, P<0.01) at baseline. All three indices significantly increased with age, and CK and KISA values were associated with a positive family history for keratoconus (P<0.001 for CK and P=0.05 for KISA), however, the two groups were not statistically different in progression of keratoconus. After grouped unaffected relatives as the high risk (age<=30 or Central K>=47.2 or I-S >=1.2 or KISA>=60) and the low risk (age>30 and Central K<47.2 and I-S <1.2 and KISA<60), relatives in the high risk group had a greater increase in CK and I-S values than those in the low risk group (CK: p=0.009; I-S: p<0.001), which indicated that there were significantly different rates of progression between two groups.
Unaffected relatives had higher videokeratography indices than normal controls, but overall they did not progress to keratoconus quicker than normal controls. However, relatives in a high risk group may have a greater risk of progression to keratoconus.
keratoconus; progression; videokeratography
To explore the atherogenic hypothesis of uterine fibroids among Chinese women.
In a case-control study, 335 patients confirmed by ultrasound or hysterectomy surgery and 539 controls were enrolled between October 1, 2009 and April 1, 2012. Unconditional logistic regressions were used to calculate the odds ratios (ORs) for the associations of subclinical atherogenic and cardiovascular risk parameters with uterine fibroids in the overall case group and hysterectomy-confirmed case group, respectively.
Higher level of ankle-brachial index (ABI) was independently associated with increased odds of uterine fibroids. The odds of UF among women in the highest tertile of ABI were 1.88 times higher (95%CI: 1.17, 3.02, Ptrend = 0.008) compared to those in the lowest tertile. The serum concentration of homocysteine was inversely related to fibroids (middle vs. low: OR 0.56, 95%CI: 0.36, 0.85; high vs. low: OR 0.50, 95% CI: 0.32, 0.79; Ptrend = 0.002). In the hysterectomy-confirmed group, an inverse association was suggested between high-density lipoprotein cholesterol (HDL-C) and fibroids (OR 0.46, 95% CI: 0.25, 0.84, Ptrend = 0.014). Moreover, the effect of homocysteine concentration was not observed in this group.
These findings suggest that women with uterine fibroids might have an increased risk of subclinical atherosclerosis.
Mild retinopathy (microaneurysms or dot-blot hemorrhages) is observed in persons without diabetes or hypertension and may reflect microvascular disease in other organs. We conducted a genome-wide association study (GWAS) of mild retinopathy in persons without diabetes.
A working group agreed on phenotype harmonization, covariate selection and analytic plans for within-cohort GWAS. An inverse-variance weighted fixed effects meta-analysis was performed with GWAS results from six cohorts of 19,411 Caucasians. The primary analysis included individuals without diabetes and secondary analyses were stratified by hypertension status. We also singled out the results from single nucleotide polymorphisms (SNPs) previously shown to be associated with diabetes and hypertension, the two most common causes of retinopathy.
No SNPs reached genome-wide significance in the primary analysis or the secondary analysis of participants with hypertension. SNP, rs12155400, in the histone deacetylase 9 gene (HDAC9) on chromosome 7, was associated with retinopathy in analysis of participants without hypertension, −1.3±0.23 (beta ± standard error), p = 6.6×10−9. Evidence suggests this was a false positive finding. The minor allele frequency was low (∼2%), the quality of the imputation was moderate (r2 ∼0.7), and no other common variants in the HDAC9 gene were associated with the outcome. SNPs found to be associated with diabetes and hypertension in other GWAS were not associated with retinopathy in persons without diabetes or in subgroups with or without hypertension.
This GWAS of retinopathy in individuals without diabetes showed little evidence of genetic associations. Further studies are needed to identify genes associated with these signs in order to help unravel novel pathways and determinants of microvascular diseases.
Polycystic ovary syndrome (PCOS) is a complex endocrine disorder with a strong familial component. PCOS is characterized by hyperandrogenemia and irregular menses. A recent genome wide association study of PCOS in a Chinese cohort identified three reproducible PCOS susceptibility loci mapping to 2p16.3 (luteinizing hormone/choriogonadotropin receptor; LHCGR), 2p21 (thyroid associated protein; THADA), and 9q33.3 (DENN/MADD domain containing 1A; DENNDIA). The impact of these loci in non-Chinese PCOS cohorts remains to be determined.
We tested association with PCOS of seven single nucleotide polymorphisms mapping to the three Chinese PCOS loci in two European-derived PCOS cohorts (Cohort A = 939 cases and 957 controls; Cohort B = 535 cases and 845 controls). Cases fulfilled the NICHD criteria for PCOS. Variation in DENND1A was strongly associated with PCOS in our cohort (pcombined cohorts=10−8 ); multiple variants in THADA were also associated with PCOS, while there was no significant evidence for association of LHCGR variation with PCOS. We had greater than 80% power to detect an effect of similar size as was observed by Chen et al. for DENND1A and THADA but reduced power (at <40%) for LHCGR at p=0.0001. We had sufficient power (57-88%) for LHCGR at p=0.01.
At least two of the PCOS susceptibility loci identified in the Chinese PCOS GWAS (DENND1A and THADA) are also associated with PCOS in European-derived populations, and therefore likely to be important in the etiology of PCOS regardless of ethnicity. Our analysis of the LHCGR gene was not sufficiently powered to detect modest effects.
PCOS; DENND1A; THADA; genome-wide association study
Keratoconus is a condition in which the cornea progressively thins over time, and is a major cause for cornea transplantation. To identify keratoconus susceptibility regions, we performed a comprehensive genome-wide association study (GWAS) using a discovery and replication design. A discovery panel of 222 keratoconus Caucasian patients and 3324 Caucasian controls was genotyped using Illumina 370K beadchips. Further associated and fine-mapping single nucleotide polymorphisms (SNPs) (n= 4905) were genotyped in an independent replication case–control panel of 304 cases and 518 controls and a family panel of 307 subjects in 70 families. Logistic regression models implemented in PLINK were performed to test associations in case–control samples with and without principal component (PC) adjustments. Generalized estimation equation models accounting for familial correlations implemented in GWAF were used for association testing in families. No genome-wide associations were identified in the discovery GWAS panel. From the initial testing without adjustments for PCs, the top three SNPs located at 3p26 (rs6442925), 2q21.3 (rs4954218) and 19q13.3 (rs1428642) were identified with unadjusted P-values of 6.5 × 10−8, 2.4 × 10−7 and 3.1 × 10−7, respectively. After adjustments for PCs, rs1428642 became the most significant through the genome with a P-value of 1.4 × 10−6, while rs6442925 and rs4954218 were less significant (P= 1.9 × 10−5 and 2.6 × 10−4). SNP rs4954218 was confirmed in two independent replication panels with P-values of 0.004 and 0.009, respectively. Meta-analysis revealed a highest association at rs4954218 with adjusted P= 1.6 × 10−7 (unadjusted P= 1.2 × 10−9). These findings suggest SNP rs4954218, located near the RAB3GAP1 gene, previously reported to be associated with corneal malformation, is a potential susceptibility locus for keratoconus.
Genetic factors explain a majority of risk variance for age-related macular degeneration (AMD). While genome-wide association studies (GWAS) for late AMD implicate genes in complement, inflammatory and lipid pathways, the genetic architecture of early AMD has been relatively under studied. We conducted a GWAS meta-analysis of early AMD, including 4,089 individuals with prevalent signs of early AMD (soft drusen and/or retinal pigment epithelial changes) and 20,453 individuals without these signs. For various published late AMD risk loci, we also compared effect sizes between early and late AMD using an additional 484 individuals with prevalent late AMD. GWAS meta-analysis confirmed previously reported association of variants at the complement factor H (CFH) (peak P = 1.5×10−31) and age-related maculopathy susceptibility 2 (ARMS2) (P = 4.3×10−24) loci, and suggested Apolipoprotein E (ApoE) polymorphisms (rs2075650; P = 1.1×10−6) associated with early AMD. Other possible loci that did not reach GWAS significance included variants in the zinc finger protein gene GLI3 (rs2049622; P = 8.9×10−6) and upstream of GLI2 (rs6721654; P = 6.5×10−6), encoding retinal Sonic hedgehog signalling regulators, and in the tyrosinase (TYR) gene (rs621313; P = 3.5×10−6), involved in melanin biosynthesis. For a range of published, late AMD risk loci, estimated effect sizes were significantly lower for early than late AMD. This study confirms the involvement of multiple established AMD risk variants in early AMD, but suggests weaker genetic effects on the risk of early AMD relative to late AMD. Several biological processes were suggested to be potentially specific for early AMD, including pathways regulating RPE cell melanin content and signalling pathways potentially involved in retinal regeneration, generating hypotheses for further investigation.
Brachypodium distachyon L. is a newly emerging model plant system for temperate cereal crop species. However, its grain protein compositions are still not clear. In the current study, we carried out a detailed proteomics and molecular genetics study on grain glutenin proteins in B. distachyon.
SDS-PAGE and RP-HPLC analysis of grain proteins showed that Brachypodium has few gliadins and high molecular weight glutenin subunits. In contrast the electrophoretic patterns for the albumin, globulin and low molecular weight glutenin subunit (LMW-GS) fractions of the grain protein were similar to those in wheat. In particular, the LMW-C type subunits in Brachypodium were more abundant than the equivalent proteins in common wheat. Southern blotting analysis confirmed that Brachypodium has 4–5 copies of LMW-GS genes. A total of 18 LMW-GS genes were cloned from Brachypodium by allele specific PCR. LMW-GS and 4 deduced amino acid sequences were further confirmed by using Western-blotting and MALDI-TOF-MS. Phylogenetic analysis indicated that Brachypodium was closer to Ae. markgrafii and Ae. umbellulata than to T. aestivum.
Brachypodium possessed a highly conserved Glu-3 locus that is closely related to Triticum and related species. The presence of LMW-GS in B. distachyon grains indicates that B. distachyon may be used as a model system for studying wheat quality attributes.
The analyses of protein synthesis, accumulation and regulation during grain development in wheat are more complex because of its larger genome size compared to model plants such as Arabidopsis and rice. In this study, grains from two wheat cultivars Jimai 20 and Zhoumai 16 with different gluten quality properties were harvested at five development stages, and were used to displayed variable expression patterns of grain proteins.
Proteome characterization during grain development in Chinese bread wheat cultivars Jimai 20 and Zhoumai 16 with different quality properties was investigated by 2-DE and tandem MALDI-TOF/TOF-MS. Identification of 117 differentially accumulated protein spots representing 82 unique proteins and five main expression patterns enabled a chronological description of wheat grain formation. Significant proteome expression differences between the two cultivars were found; these included 14 protein spots that accumulated in both cultivars but with different patterns and 27 cultivar-different spots. Among the cultivar-different protein spots, 14 accumulated in higher abundance in Jimai 20 than in Zhoumai 16, and included NAD-dependent isocitrate dehydrogenase, triticin precursor, LMW-s glutenin subunit and replication factor C-like protein. These proteins are likely to be associated with superior gluten quality. In addition, some proteins such as class II chitinase and peroxidase 1 with isoforms in developing grains were shown to be phosphorylated by Pro-Q Diamond staining and phosphorprotein site prediction. Phosphorylation could have important roles in wheat grain development. qRT-PCR analysis demonstrated that transcriptional and translational expression patterns of many genes were significantly different.
Wheat grain proteins displayed variable expression patterns at different developmental stages and a considerable number of protein spots showed differential accumulation between two cultivars. Differences in seed storage proteins were considered to be related to different quality performance of the flour from these wheat cultivars. Some proteins with isoforms were phosphorylated, and this may reflect their importance in grain development. Our results provide new insights into proteome characterization during grain development in different wheat genotypes.
Wheat; Grain proteome; Phosphorproteins; 2-DE; Tandem MS; qRT-PCR
The growth process of Bacillus thuringiensis Bt4.0718 strain was studied using proteomic technologies. The proteins of Bt whole cells at three phases—middle vegetative, early sporulation, and late sporulation—were extracted with lysis buffer, followed with separation by 2-DE and identified by MALDI-TOF/TOF MS. Bioactive factors such as insecticidal crystal proteins (ICPs) including Cry1Ac(3), Cry2Aa, and BTRX28, immune inhibitor (InhA), and InhA precursor were identified. InhA started to express at the middle vegetative phase, suggesting its contribution to the survival of Bt in the host body. At the early sporulation phase, ICPs started their expression. CotJC, OppA, ORF1, and SpoIVA related to the formation of crystals and spores were identified, the expression characteristics of which ensured the stable formation of crystals and spores. This study provides an important foundation for further exploration of the stable expression of ICPs, the smooth formation of crystals, and the construction of recombinant strains.
This is a meta-analysis of two genome-wide association studies that found evidence of association of keratoconus with polymorphisms in the promoter of the HGF gene. One polymorphism is associated with higher levels of serum HGF.
Keratoconus is a progressive disorder of the cornea that can lead to severe visual impairment or blindness. Although several genomic regions have been linked to rare familial forms of keratoconus, no genes have yet been definitively identified for common forms of the disease.
Two genome-wide association scans were undertaken in parallel. The first used pooled DNA from an Australian cohort, followed by typing of top-ranked single-nucleotide polymorphisms (SNPs) in individual DNA samples. The second was conducted in individually genotyped patients, and controls from the USA. Tag SNPs around the hepatocyte growth factor (HGF) gene were typed in three additional replication cohorts. Serum levels of HGF protein in normal individuals were assessed with ELISA and correlated with genotype.
The only SNP observed to be associated in both the pooled discovery and primary replication cohort was rs1014091, located upstream of the HGF gene. The nearby SNP rs3735520 was found to be associated in the individually typed discovery cohort (P = 6.1 × 10−7). Genotyping of tag SNPs around HGF revealed association at rs3735520 and rs17501108/rs1014091 in four of the five cohorts. Meta-analysis of all five datasets together yielded suggestive P values for rs3735520 (P = 9.9 × 10−7) and rs17501108 (P = 9.9 × 10−5). In addition, SNP rs3735520 was found to be associated with serum HGF level in normal individuals (P = 0.036).
Taken together, these results implicate genetic variation at the HGF locus with keratoconus susceptibility.
This phase 1 study assessed the safety, tolerability, pharmacokinetics, and preliminary antitumor activity of linifanib in Japanese patients with advanced solid tumors.
Patients were assigned to one of four sequential cohorts (0.05, 0.10, 0.20, or 0.25 mg/kg) of oral, once-daily linifanib on a 21-day cycle. Adverse events (AEs) were assessed per common terminology criteria for adverse events v3.0; tumor responses were assessed by response evaluation criteria in solid tumors.
Eighteen patients were enrolled. Eleven (61%) received ≥3 prior therapies. Dose-limiting toxicities were Grade 3 ALT increase (0.10 mg/kg linifanib) and Grade 1 T-wave inversion (0.25 mg/kg linifanib) requiring dose interruption for >7 days and discontinuation on day 29. The most common linifanib-related AE was hypertension. Other significant treatment-related AEs included proteinuria, fatigue, and palmar-plantar erythrodysaesthesia. Linifanib pharmacokinetics were dose-proportional across 0.10–0.25 mg/kg. Two patients (11.1%) had confirmed partial responses, 12 had a best response of stable disease (11 had stable disease for ≥12 weeks), and four patients were not evaluable due to incomplete data. Four patients (lung cancer, breast cancer, thymic cancer, sarcoma) have continued linifanib for ≥48 weeks (range, 48–96+ weeks).
Linifanib was well tolerated with promising preliminary clinical activity in Japanese patients. Later-phase global studies examining linifanib efficacy will include Japanese patients.
Angiogenesis; Linifanib (ABT-869); PDGFR; Solid tumors; VEGFR; Japanese
Piceatannol (3,3′,4,5′-tetrahydroxy-trans-stilbene; Pice), found in a variety of plant sources including grapes, red wine, peanuts and rhubarb, is known as a metabolite and analog of Resveratrol (3,5,4′-trihydroxy-trans-stilbene; Res) and has higher bioactivity than Res. To explore the mechanism of DNA damage induced by Pice in the presence of copper (Cu)(II), gel electrophoresis, UV-visible spectroscopy, fluorescence spectroscopy and Fourier transform infrared spectroscopy were used. The results of gel electrophoresis demonstrated that the hydroxyl radical played a critical role in DNA cleavage. Spectroscopy confirmed that the mechanism of DNA cleavage induced by Pice-Cu(II) involves the Haber Weiss and Fenton reactions. Pice chelates with Cu(II) as a bidentate ligand, and the Pice-Cu(II) complex undergoes intramolecular electron transfer to form the semiquinone radical anion and Cu(I), which may be reoxidated by O2 to form Cu(II) with hydroxyl radical generation. In brief, the formation of the hydroxyl radical and the Cu(II)/Cu(I) redox cycle play a key role in inducing DNA damage. In this process, Pice demonstrated pro-oxidant properties. Oxidative product(s) of Pice, semiquinone, was formed and Cu(I) was reoxidized to Cu(II). The redox cycling of copper generated reactive oxygen species, which induced DNA cleavage, the hallmark of cell apoptosis. The mechanism of DNA breakage induced by Pice-Cu(II) may be a significant pathway through which cancer cells are killed.
piceatannol; anticancer; pro-oxidant; polyphenols; copper
Genetic association studies usually involve a large number of single-nucleotide polymorphisms (SNPs) (k) and a relative small sample size (n), which produces the situation that k is much greater than n. Because conventional statistical approaches are unable to deal with multiple SNPs simultaneously when k is much greater than n, single-SNP association studies have been used to identify genes involved in a disease’s pathophysiology, which causes a multiple testing problem. To evaluate the contribution of multiple SNPs simultaneously to disease traits when k is much greater than n, we developed the Bayesian regression with singular value decomposition (BRSVD) method. The method reduces the dimension of the design matrix from k to n by applying singular value decomposition to the design matrix. We evaluated the model using a Markov chain Monte Carlo simulation with Gibbs sampler constructed from the posterior densities driven by conjugate prior densities. Permutation was incorporated to generate empirical p-values. We applied the BRSVD method to the sequence data provided by Genetic Analysis Workshop 17 and found that the BRSVD method is a practical method that can be used to analyze sequence data in comparison to the single-SNP association test and the penalized regression method.
Novel pathways in polycystic ovary syndrome (PCOS) are being identified in gene expression studies in PCOS tissues; such pathways may contain key genes in disease etiology. Previous expression studies identified both dickkopf homolog 1 (DKK1) and DnaJ (Hsp40) homolog, subfamily B, member 1 (DNAJB1) as differentially expressed in PCOS tissue, implicating them as candidates for PCOS susceptibility. To test this, we genotyped a discovery cohort of 335 PCOS cases and 198 healthy controls for three DKK1 single nucleotide polymorphisms (SNPs) and four DNAJB1 SNPs and a replication cohort of 396 PCOS cases and 306 healthy controls for 1 DKK1 SNP and 1 DNAJB1 SNP. SNPs and haplotypes were determined and tested for association with PCOS and component phenotypes. We found that no single nucleotide polymorphisms were associated with PCOS risk; however, the major allele of rs1569198 from DKK1 was associated with increased total testosterone (discovery cohort P = 0.0035) and dehydroepiandrosterone sulfate (replication cohort P = 0.05). Minor allele carriers at rs3962158 from DNAJB1 had increased fasting insulin (discovery cohort P = 0.003), increased HOMA-IR (discovery cohort P = 0.006; replication cohort P = 0.036), and increased HOMA-%B (discovery cohort P = 0.004). Carriers of haplotype 2 at DNAJB1 also had increased fasting insulin, HOMA-IR, and HOMA-%B. These findings suggest that genetic variation in DKK1 and DNAJB1 may have a role in the hyperandrogenic and metabolic dysfunction of PCOS, respectively. Our results also demonstrate the utility of gene expression data as a source of novel candidate genes in PCOS, a complex and still incompletely defined disease, for which alternative methods of gene identification are needed.
Serum concentrations of total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) are among the most important risk factors for coronary artery disease (CAD) and are targets for therapeutic intervention. We screened the genome for common variants associated with serum lipids in >100,000 individuals of European ancestry. Here we report 95 significantly associated loci (P < 5 × 10-8), with 59 showing genome-wide significant association with lipid traits for the first time. The newly reported associations include single nucleotide polymorphisms (SNPs) near known lipid regulators (e.g., CYP7A1, NPC1L1, and SCARB1) as well as in scores of loci not previously implicated in lipoprotein metabolism. The 95 loci contribute not only to normal variation in lipid traits but also to extreme lipid phenotypes and impact lipid traits in three non-European populations (East Asians, South Asians, and African Americans). Our results identify several novel loci associated with serum lipids that are also associated with CAD. Finally, we validated three of the novel genes—GALNT2, PPP1R3B, and TTC39B—with experiments in mouse models. Taken together, our findings provide the foundation to develop a broader biological understanding of lipoprotein metabolism and to identify new therapeutic opportunities for the prevention of CAD.
There is increasing evidence that the microcirculation plays an important role in the pathogenesis of cardiovascular diseases. Changes in retinal vascular caliber reflect early microvascular disease and predict incident cardiovascular events. We performed a genome-wide association study to identify genetic variants associated with retinal vascular caliber. We analyzed data from four population-based discovery cohorts with 15,358 unrelated Caucasian individuals, who are members of the Cohort for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium, and replicated findings in four independent Caucasian cohorts (n = 6,652). All participants had retinal photography and retinal arteriolar and venular caliber measured from computer software. In the discovery cohorts, 179 single nucleotide polymorphisms (SNP) spread across five loci were significantly associated (p<5.0×10−8) with retinal venular caliber, but none showed association with arteriolar caliber. Collectively, these five loci explain 1.0%–3.2% of the variation in retinal venular caliber. Four out of these five loci were confirmed in independent replication samples. In the combined analyses, the top SNPs at each locus were: rs2287921 (19q13; p = 1.61×10−25, within the RASIP1 locus), rs225717 (6q24; p = 1.25×10−16, adjacent to the VTA1 and NMBR loci), rs10774625 (12q24; p = 2.15×10−13, in the region of ATXN2,SH2B3 and PTPN11 loci), and rs17421627 (5q14; p = 7.32×10−16, adjacent to the MEF2C locus). In two independent samples, locus 12q24 was also associated with coronary heart disease and hypertension. Our population-based genome-wide association study demonstrates four novel loci associated with retinal venular caliber, an endophenotype of the microcirculation associated with clinical cardiovascular disease. These data provide further insights into the contribution and biological mechanisms of microcirculatory changes that underlie cardiovascular disease.
The microcirculation plays an important role in the development of cardiovascular diseases. Retinal vascular caliber changes reflect early microvascular disease and predict incident cardiovascular events. In order to identify genetic variants associated with retinal vascular caliber, we performed a genome-wide association study and analyzed data from four population-based discovery cohorts with 15,358 unrelated Caucasian individuals, who are members of the Cohort for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium, and replicated findings in four independent Caucasian cohorts (n = 6,652). We found evidence for association of four loci with retinal venular caliber: on chromosomes 19q13 within the RASIP1 locus, 6q24 adjacent to the VTA1 and NMBR loci, 12q24 in the region of ATXN2,SH2B3 and PTPN11 loci, and 5q14 adjacent to the MEF2C locus. In two independent samples, locus 12q24 was also associated with coronary heart disease and hypertension. In the present study, we demonstrate that four novel loci were associated with retinal venular caliber, an endophenotype of the microcirculation associated with clinical cardiovascular disease. Our findings will help focus research on novel genes and pathways involving the microcirculation and its role in the development of cardiovascular disease.
HMG-CoA reductase (HMGCR) is an enzyme involved in cholesterol synthesis. To investigate the contribution of the HMGCR gene to lipids and lipoprotein subfraction in different ethnicities, we performed an association study in the Multi-Ethnic Study of Atherosclerosis (MESA). Totally, 2444 MESA subjects (597 African-Americans (AA), 627 Chinese-Americans (CHA), 612 European-Americans (EA), and 608 Hispanic-Americans (HA)) without statin use were included. Participants had measurements of blood pressure, anthropometry, and fasting blood samples. Subjects were genotyped for 10 single nucleotide polymorphisms (SNPs). After excluding SNPs with minor allele frequency <5%, a single block was constructed. The most frequent haplotype was H1 (41-56%) in all ethnic groups except AA (H2a, 44.9%). Lower triglyceride level was associated with the H2a haplotype in AA and H2 in HA. In HA, H4 carriers had higher levels of triglyceride and small low-density lipoprotein (s-LDL), and lower high-density lipoprotein cholesterol (HDL-c), while carriers with H7 or H8 had associations with these traits in the opposite direction. No significant association was discovered in both CHA and EA. The total variation for triglyceride that could be explained by H2 alone was 2.6% in HA and 1.4% in AA. In conclusion, HMGCR gene variation is associated with multiple lipid/lipoprotein traits, especially with triglyceride, s-LDL, and HDL-c. The impact of the genetic variance is modest and differs greatly between ethnicities.
hydroxymethylglutaryl CoA reductases; association study; cholesterol; triglyceride; low density lipoprotein size
Statins effectively lower total and plasma LDL-cholesterol, but the magnitude of decrease varies among individuals. To identify single nucleotide polymorphisms (SNPs) contributing to this variation, we performed a combined analysis of genome-wide association (GWA) results from three trials of statin efficacy.
Methods and Principal Findings
Bayesian and standard frequentist association analyses were performed on untreated and statin-mediated changes in LDL-cholesterol, total cholesterol, HDL-cholesterol, and triglyceride on a total of 3932 subjects using data from three studies: Cholesterol and Pharmacogenetics (40 mg/day simvastatin, 6 weeks), Pravastatin/Inflammation CRP Evaluation (40 mg/day pravastatin, 24 weeks), and Treating to New Targets (10 mg/day atorvastatin, 8 weeks). Genotype imputation was used to maximize genomic coverage and to combine information across studies. Phenotypes were normalized within each study to account for systematic differences among studies, and fixed-effects combined analysis of the combined sample were performed to detect consistent effects across studies. Two SNP associations were assessed as having posterior probability greater than 50%, indicating that they were more likely than not to be genuinely associated with statin-mediated lipid response. SNP rs8014194, located within the CLMN gene on chromosome 14, was strongly associated with statin-mediated change in total cholesterol with an 84% probability by Bayesian analysis, and a p-value exceeding conventional levels of genome-wide significance by frequentist analysis (P = 1.8×10−8). This SNP was less significantly associated with change in LDL-cholesterol (posterior probability = 0.16, P = 4.0×10−6). Bayesian analysis also assigned a 51% probability that rs4420638, located in APOC1 and near APOE, was associated with change in LDL-cholesterol.
Conclusions and Significance
Using combined GWA analysis from three clinical trials involving nearly 4,000 individuals treated with simvastatin, pravastatin, or atorvastatin, we have identified SNPs that may be associated with variation in the magnitude of statin-mediated reduction in total and LDL-cholesterol, including one in the CLMN gene for which statistical evidence for association exceeds conventional levels of genome-wide significance.
PRINCE and TNT are not registered. CAP is registered at Clinicaltrials.gov NCT00451828
We performed association analysis under a previous linkage peak on chromosome 16 with genome-wide single-nucleotide polymorphism (SNP) data to identify genetic variants underlying body mass index (BMI). Data from all subjects with baseline measures and a subgroup who had complete data at four selected time points from the Framingham Heart Study were analyzed. The cross-sectional measures include BMI at baseline for all subjects, as well as BMI at selected time points for the subgroup. The longitudinal measure is the within-subject mean of BMI for the subgroup at the four time points.
Association analysis was first performed using PLINK after dividing large pedigrees into nuclear families. We then followed up the identified regions by variance-components methods as implemented in SOLAR using the extended pedigrees.
The strongest evidence for associations were observed at 52.3 Mbp (PLINK p = 0.00002, QTLD p = 0.005), on the FTO gene, and at 48.1 Mbp (PLINK p = 0.002, QTLD p = 0.0006) on chromosome 16, which are directly under the previous identified linkage peak. This association was consistently observed for all samples at baseline, and for the subgroup at time point 2, 3, 4 and MEAN, both by PLINK and SOLAR. In addition, another SNP/region at 46.7 Mbp on same chromosome was found to be associated with several BMI measures in the subgroup. Fine-mapping with more markers provided further evidence for SNP association with BMI in the same region (at 52.4 Mbp, QTLD p = 0.0003).
These results suggest the existence of genes/DNA variations in these regions that contribute to BMI variation.
The transforming growth factor β (TGFβ) family has critical roles in the regulation of fertility. In addition, the pathogenesis of some human cancers is attributed to misregulation of TGFβ function and SMAD2 or SMAD4 mutations. There are limited mouse models for the BMP signaling SMADs (BR-SMADs) 1, 5, and 8 because of embryonic lethality and suspected genetic redundancy. Using tissue-specific ablation in mice, we deleted the BR-SMADs from somatic cells of ovaries and testes. Single conditional knockouts for Smad1 or Smad5 or mice homozygous null for Smad8 are viable and fertile. Female double Smad1 Smad5 and triple Smad1 Smad5 Smad8 conditional knockout mice become infertile and develop metastatic granulosa cell tumors. Male double Smad1 Smad5 conditional knockout mice are fertile but demonstrate metastatic testicular tumor development. Microarray analysis indicated significant alterations in expression of genes related to the TGFβ pathway, as well as genes involved in infertility and extracellular matrix production. These data strongly implicate the BR-SMADs as part of a critical developmental pathway in ovaries and testis that, when disrupted, leads to malignant transformation.
We examined the potential gene × gene interactions and gene × smoking interactions in rheumatoid arthritis (RA) using the candidate gene data sets provided by Genetic Analysis Workshop 15 Problem 2. The multifactor dimensionality reduction (MDR) method was used to test gene × gene interactions among candidate genes. The case-only sample was used to test gene × smoking interactions. The best predictive model was the single-locus model with single-nucleotide polymorphism (SNP) rs2476601 in gene PTPN22. However, no clear gene × gene interaction was identified. Substantial departure from multiplicativity was observed between smoking and SNPs in genes CTLA4, PADI4, MIF, and SNPs on chromosome 5 and one haplotype of PTPN22. The strongest evidence of association was identified between the PTPN22 gene and RA status, which was consistently detected in single SNP association, gene × gene interaction and gene × smoking interaction analyses.
To evaluate linkage evidence for body mass index (BMI) using both cross-sectional and longitudinal data, we performed genome-wide multipoint linkage analyses on subjects who had complete data at four selected time points (initial, 8th, 12th, and 16th year following the initial visit) from the Framingham Heart Study. The cross-sectional measures included BMI at each of the four selected time points and the longitudinal measure was the within-subject mean of BMI at the above four time points.
Using the variance components method, we consistently observed the maximum LOD score out of the genome scan using BMI at each time point and the mean of BMI between 049xd2 and GATA71H05 on chromosome 16. The highest LOD score (3.0) was at time point 1, while the lowest (1.9) was at time point 4. We also observed other suggestive linkages on chromosome 6, 10, and 18 at time point 1 only.
The longitudinal measure we studied (mean of BMI) did not provide greater power to identify a positive linkage than some of the cross-sectional measures (e.g., time point 1). The changing of linkage evidence over time provided some insights on the variation of genetic effect on BMI with aging. There may be a QTL on chromosome 16 that contributes to BMI and this locus, and maybe others, is more likely to affect BMI during early adulthood.
The Framingham Heart Study is a very successful longitudinal research for cardiovascular diseases. The completion of a 10-cM genome scan in Framingham families provided an opportunity to evaluate linkage using longitudinal data. Several descriptive traits based on simulated longitudinal data from the Genetic Analysis Workshop 13 (GAW13) were generated, and linkage analyses were performed for these traits. We compared the power of detecting linkage for baseline and slope genes in the simulated data of GAW13 using these traits. We found that using longitudinal traits based on multiple follow-ups may not be more powerful than using cross-sectional traits for genetic linkage analysis.