Complex trait genome-wide association studies (GWAS) provide an efficient strategy for evaluating large numbers of common variants in large numbers of individuals and for identifying trait-associated variants. Nevertheless, GWAS often leave much of the trait heritability unexplained. We hypothesized that some of this unexplained heritability might be due to common and rare variants that reside in GWAS identified loci but lack appropriate proxies in modern genotyping arrays. To assess this hypothesis, we re-examined 7 genes (APOE, APOC1, APOC2, SORT1, LDLR, APOB, and PCSK9) in 5 loci associated with low-density lipoprotein cholesterol (LDL-C) in multiple GWAS. For each gene, we first catalogued genetic variation by re-sequencing 256 Sardinian individuals with extreme LDL-C values. Next, we genotyped variants identified by us and by the 1000 Genomes Project (totaling 3,277 SNPs) in 5,524 volunteers. We found that in one locus (PCSK9) the GWAS signal could be explained by a previously described low-frequency variant and that in three loci (PCSK9, APOE, and LDLR) there were additional variants independently associated with LDL-C, including a novel and rare LDLR variant that seems specific to Sardinians. Overall, this more detailed assessment of SNP variation in these loci increased estimates of the heritability of LDL-C accounted for by these genes from 3.1% to 6.5%. All association signals and the heritability estimates were successfully confirmed in a sample of ∼10,000 Finnish and Norwegian individuals. Our results thus suggest that focusing on variants accessible via GWAS can lead to clear underestimates of the trait heritability explained by a set of loci. Further, our results suggest that, as prelude to large-scale sequencing efforts, targeted re-sequencing efforts paired with large-scale genotyping will increase estimates of complex trait heritability explained by known loci.

Our data suggest that a genetic approach could be useful in providing molecular support to the hypothesis that hypersensitivity of the dopaminergic system may represent the pathophysiologic basis of migraine, at least in a subgroup of patients.

Androgenetic alopecia (AGA) is a common heritable polygenic disorder whose genetics is not fully understood, even though it seems to be X-linked. We carried out an epidemiological survey for AGA on 9,000 people from 8 isolated villages of a secluded region of Sardinia (Ogliastra), and identified a large cohort of affected individuals. We genotyped 200 cases and 200 controls (mean kinship 0.001) with the 500k chip array and conducted case-control association analysis on the X chromosome. We identified Xq11-q12 as strongly associated with AGA. In particular, we found that rs1352015 located 8 kb from the EDA2R gene showed the best result (P=7.77e(-7)). This region also contains the AR gene, hence we tested both genes in 492 cases and 492 controls. We found that the non-synonymous SNP rs1385699 on EDA2R gave the best result (P=3.9e(-19)) whereas rs6152 on the AR gene is less significant (P=4.17e(-12)). Further statistical analysis carried out by conditioning each gene to the presence of the other showed that the association with EDA2R is independent while the association with AR seems to be the result of linkage disequilibrium. These results give insight into the pathways involved in AGA etiology.

No genes influencing oculometric phenotypes have yet been identified, despite it being well known that eye morphometry is involved in refraction and that genetics may play an important role. We have therefore performed a heritability analysis and genome-wide search (GWS) of biometric ocular traits in an isolated Sardinian population, assessing the genetic contribution and identifying the associated genetic loci. A complete eye examination including refraction and ocular biometry measurements such as axial length (AL), anterior chamber depth (ACD) and corneal curvature (CC), was performed on 789 subjects. Heritability analysis was carried out by means of parent-offspring regression and variance component models. Univariate and bivariate linkage analysis was performed by using 654 microsatellite markers spanning the genome. CC showed a mean heritability of 57%. AL and ACD were found to have significantly different variances (P<0.01) in males and females, so that heritability was calculated separately for each sex. AL had an estimated heritability in females of 31% and in males of 60%, whereas ACD had an estimated heritability of 47% in females and of 44% in males. In the GWS, the most suggestive evidence of linkage was identified on chromosome 2 for AL (LOD 2.64), on chromosome 1 for ACD (LOD 2.32) and on chromosomes 7, 2 and 3 for CC (LOD 2.50, 2.44 and 2.34, respectively). High heritability of eye morphometry traits was thus revealed. The identified loci are the first linkage signals available in ocular biometry. Notably, the observed significant differences in parental transmission deserve further study.

BackgroundAlzheimer's disease is the most common progressive neurodegenerative disease. In recent years, numerous progresses in the discovery of novel Alzheimer's disease molecular biomarkers in brain as well as in biological fluids have been made. Among them, those involving lipid metabolism are emerging as potential candidates. In particular, an accumulation of neutral lipids was recently found by us in skin fibroblasts from Alzheimer's disease patients. Therefore, with the aim to assess whether peripheral alterations in cholesterol homeostasis might be relevant in Alzheimer's disease development and progression, in the present study we analyzed lipid metabolism in plasma and peripheral blood mononuclear cells from Alzheimer's disease patients and from their first-degree relatives.MethodsBlood samples were obtained from 93 patients with probable Alzheimer's disease and from 91 of their first-degree relatives. As controls we utilized 57, cognitively normal, over-65 year-old volunteers and 113 blood donors aged 21-66 years, respectively. Data are reported as mean ± standard error. Statistical calculations were performed using the statistical analysis software Origin 8.0 version. Data analysis was done using the Student t-test and the Pearson test.ResultsData reported here show high neutral lipid levels and increased ACAT-1 protein in about 85% of peripheral blood mononuclear cells freshly isolated (ex vivo) from patients with probable sporadic Alzheimer's disease compared to about 7% of cognitively normal age-matched controls. A significant reduction in high density lipoprotein-cholesterol levels in plasma from Alzheimer's disease blood samples was also observed. Additionally, correlation analyses reveal a negative correlation between high density lipoprotein-cholesterol and cognitive capacity, as determined by Mini Mental State Examination, as well as between high density lipoprotein-cholesterol and neutral lipid accumulation. We observed great variability in the neutral lipid-peripheral blood mononuclear cells data and in plasma lipid analysis of the subjects enrolled as Alzheimer's disease-first-degree relatives. However, about 30% of them tend to display a peripheral metabolic cholesterol pattern similar to that exhibited by Alzheimer's disease patients.ConclusionWe suggest that neutral lipid-peripheral blood mononuclear cells and plasma high density lipoprotein-cholesterol determinations might be of interest to outline a distinctive metabolic profile applying to both Alzheimer's disease patients and asymptomatic subjects at higher risk of disease.

Recently, a major locus on chromosome 7q was found in association with the taste sensitivity to phenylthiocarbamide (PTC) in humans. This region contains the TAS2R38 gene that encodes a member of the TAS2R bitter taste receptor family. Three SNPs within this gene demonstrated a strong association with taster status in Utah families and in an additional sample of 85 unrelated individuals. We studied a small isolated village in eastern Sardinia and carried out a genome-wide scan to map the genetic basis of PTC perception in this population. We performed both qualitative and quantitative PTC-taste linkage analysis. Qualitative analysis was carried out by defining a cut-off from the bimodal distribution of the trait and classifying subjects as tasters and non-tasters (75 and 25%, respectively). Linkage analysis on 131 subjects belonging to a unique large multi-generation pedigree comprising 239 subjects confirmed significant evidence for linkage at 7q35 also in our population. Haplotype analyses of the three SNPs inside the PTC gene allowed us to identify only two haplotypes that were associated with the non-taster phenotype (80% AVI homozygous) and to taster phenotype (40% PAV homozygous and 56% PAV/AVI heterozygous). Sex, age and haplotype effect explained 77.2 % of the total variance in PTC sensitivity.

Association analysis of candidate genes may represent a strategy for clarifying the genetic components involved in bipolar disorder. Polymorphism at dopamine receptor genes DRD2, DRD4, and dopamine and serotonin transporter genes (DAT, SERT) has been used in previous association studies. Some authors have reported positive association between certain alleles and bipolar disorder, using the case-control design. In this family-based association study of DRD2, DRD4, DAT, and SERT, the distribution of parental nontransmitted alleles was compared with that of alleles transmitted to 53 Sardinian probands suffering from bipolar disorder. The transmission disequilibrium test (TDT) was used to detect any disproportionate transmission of alleles by heterozygous parents to affected children. No differences were found between the allele distribution of polymorphisms at DRD2, DRD4, DAT, and SERT in probands and parental nontransmitted chromosomes. TDT did not reveal any difference between transmitted and nontransmitted alleles. Our results do not support the hypothesis of a role for DRD2, DRD4, DAT, or SERT in bipolar disorder. Previously reported positive associations between DRD2 or SERT and bipolar disorder were conceivably due to stratification dependent on the case-control design, even though our sample might have failed to detect small associations due to limited power.

A recent study reported a possible association between allele 1 of the dopamine D3 receptor gene and bipolar affective disorder using the haplotype relative risk approach. In attempt to replicate these findings, we used similar family-based methods, such as the Haplotype-Based Haplotype Relative Risk method and the Transmission Disequilibrium Test, in a sample of 44 bipolar probands from Sardinia with both parents available. Using the Bal I restriction enzyme site polymorphism of Lannfelt et al. (1992), no differences were found between transmitted and non-transmitted alleles and no evidence of linkage disequilibrium was observed.