Unnur Thorsteinsdottir

Identification of low-frequency and rare sequence variants associated with elevated or reduced risk of type 2 diabetes.

Through whole-genome sequencing of 2,630 Icelanders and imputation into 11,114 Icelandic cases and 267,140 controls followed by testing in Danish and Iranian samples, we discovered 4 previously unreported variants affecting risk of type 2 diabetes (T2D). A low-frequency (1.47%) variant in intron 1 of CCND2, rs76895963[G], reduces risk of T2D by half (odds ratio (OR) = 0.53, P = 5.0 × 10−21) and is correlated with increased CCND2 expression. Notably, this variant is also associated with both greater height and higher body mass index (1.17 cm per allele, P = 5.5 × 10−12 and 0.56 kg/m2 per allele, P = 6.5 × 10−7, respectively). In addition, two missense variants in PAM, encoding p.Asp563Gly (frequency of 4.98%) and p.Ser539Trp (frequency of 0.65%), confer moderately higher risk of T2D (OR = 1.23, P = 3.9 × 10−10 and OR = 1.47, P = 1.7 × 10−5, respectively), and a rare (0.20%) frameshift variant in PDX1, encoding p.Gly218Alafs*12, associates with high risk of T2D (OR = 2.27, P = 7.3 × 10−7).

The impact of low-frequency and rare variants on lipid levels

Using a genome-wide screen of 9.6 million genetic variants achieved through 1000 Genomes Project imputation in 62,166 samples, we identify association to lipid traits in 93 loci, including 79 previously identified loci with new lead SNPs and 10 new loci, 15 loci with a low-frequency lead SNP and 10 loci with a missense lead SNP, and 2 loci with an accumulation of rare variants. In six loci, SNPs with established function in lipid genetics (CELSR2, GCKR, LIPC and APOE) or candidate missense mutations with predicted damaging function (CD300LG and TM6SF2) explained the locus associations. The low-frequency variants increased the proportion of variance explained, particularly for low-density lipoprotein cholesterol and total cholesterol. Altogether, our results highlight the impact of low-frequency variants in complex traits and show that imputation offers a cost-effective alternative to resequencing.

Discovery and fine-mapping of glycaemic and obesity-related trait loci using high-density imputation

Reference panels from the 1000 Genomes (1000G) Project Consortium provide near complete coverage of common and low-frequency genetic variation with minor allele frequency ≥0.5% across European ancestry populations. Within the European Network for Genetic and Genomic Epidemiology (ENGAGE) Consortium, we have undertaken the first large-scale meta-analysis of genome-wide association studies (GWAS), supplemented by 1000G imputation, for four quantitative glycaemic and obesity-related traits, in up to 87,048 individuals of European ancestry. We identified two loci for body mass index (BMI) at genome-wide significance, and two for fasting glucose (FG), none of which has been previously reported in larger meta-analysis efforts to combine GWAS of European ancestry. Through conditional analysis, we also detected multiple distinct signals of association mapping to established loci for waist-hip ratio adjusted for BMI (RSPO3) and FG (GCK and G6PC2). The index variant for one association signal at the G6PC2 locus is a low-frequency coding allele, H177Y, which has recently been demonstrated to have a functional role in glucose regulation. Fine-mapping analyses revealed that the non-coding variants most likely to drive association signals at established and novel loci were enriched for overlap with enhancer elements, which for FG mapped to promoter and transcription factor binding sites in pancreatic islets, in particular. Our study demonstrates that 1000G imputation and genetic fine-mapping of common and low-frequency variant association signals at GWAS loci, integrated with genomic annotation in relevant tissues, can provide insight into the functional and regulatory mechanisms through which their effects on glycaemic and obesity-related traits are mediated.

Selection against variants in the genome associated with educational attainment

Significance Epidemiological studies suggest that educational attainment is affected by genetic variants. Results from recent genetic studies allow us to construct a score from a person’s genotypes that captures a portion of this genetic component. Using data from Iceland that include a substantial fraction of the population we show that individuals with high scores tend to have fewer children, mainly because they have children later in life. Consequently, the average score has been decreasing over time in the population. The rate of decrease is small per generation but marked on an evolutionary timescale. Another important observation is that the association between the score and fertility remains highly significant after adjusting for the educational attainment of the individuals. Epidemiological and genetic association studies show that genetics play an important role in the attainment of education. Here, we investigate the effect of this genetic component on the reproductive history of 109,120 Icelanders and the consequent impact on the gene pool over time. We show that an educational attainment polygenic score, POLYEDU, constructed from results of a recent study is associated with delayed reproduction (P < 10−100) and fewer children overall. The effect is stronger for women and remains highly significant after adjusting for educational attainment. Based on 129,808 Icelanders born between 1910 and 1990, we find that the average POLYEDU has been declining at a rate of ∼0.010 standard units per decade, which is substantial on an evolutionary timescale. Most importantly, because POLYEDU only captures a fraction of the overall underlying genetic component the latter could be declining at a rate that is two to three times faster.

A rare IL33 loss-of-function mutation reduces blood eosinophil counts and protects from asthma

IL-33 is a tissue-derived cytokine that induces and amplifies eosinophilic inflammation and has emerged as a promising new drug target for asthma and allergic disease. Common variants at IL33 and IL1RL1, encoding the IL-33 receptor ST2, associate with eosinophil counts and asthma. Through whole-genome sequencing and imputation into the Icelandic population, we found a rare variant in IL33 (NM_001199640:exon7:c.487-1G>C (rs146597587-C), allele frequency = 0.65%) that disrupts a canonical splice acceptor site before the last coding exon. It is also found at low frequency in European populations. rs146597587-C associates with lower eosinophil counts (β = -0.21 SD, P = 2.5×10–16, N = 103,104), and reduced risk of asthma in Europeans (OR = 0.47; 95%CI: 0.32, 0.70, P = 1.8×10–4, N cases = 6,465, N controls = 302,977). Heterozygotes have about 40% lower total IL33 mRNA expression than non-carriers and allele-specific analysis based on RNA sequencing and phased genotypes shows that only 20% of the total expression is from the mutated chromosome. In half of those transcripts the mutation causes retention of the last intron, predicted to result in a premature stop codon that leads to truncation of 66 amino acids. The truncated IL-33 has normal intracellular localization but neither binds IL-33R/ST2 nor activates ST2-expressing cells. Together these data demonstrate that rs146597587-C is a loss of function mutation and support the hypothesis that IL-33 haploinsufficiency protects against asthma.

Association analysis of 29,956 individuals confirms that a low frequency variant at CCND2 halves the risk of type 2 diabetes by enhancing insulin secretion

A recent study identified a low-frequency variant at CCND2 associated with lower risk of type 2 diabetes, enhanced insulin response to a glucose challenge, higher height, and, paradoxically, higher BMI. We aimed to replicate the strength and effect size of these associations in independent samples and to assess the underlying mechanism. We genotyped the variant in 29,956 individuals and tested its association with type 2 diabetes and related traits. The low-frequency allele was associated with a lower risk of type 2 diabetes (OR 0.53; P = 2 × 10−13; 6,647 case vs. 12,645 control subjects), higher disposition index (β = 0.07 log10; P = 2 × 10−11; n = 13,028), and higher Matsuda index of insulin sensitivity (β = 0.02 log10; P = 5 × 10−3; n = 13,118) but not fasting proinsulin (β = 0.01 log10; P = 0.5; n = 6,985). The low frequency allele was associated with higher adult height (β = 1.38 cm; P = 6 × 10−9; n = 13,927), but the association of the variant with BMI (β = 0.36 kg/m2; P = 0.02; n = 24,807), estimated in four population-based samples, was less than in the original publication where the effect estimate was biased by analyzing case subjects with type 2 diabetes and control subjects without diabetes separately. Our study establishes that a low-frequency allele in CCND2 halves the risk of type 2 diabetes primarily through enhanced insulin secretion.

A Splice Region Variant in LDLR Lowers Non-high Density Lipoprotein Cholesterol and Protects against Coronary Artery Disease

Through high coverage whole-genome sequencing and imputation of the identified variants into a large fraction of the Icelandic population, we found four independent signals in the low density lipoprotein receptor gene (LDLR) that associate with levels of non-high density lipoprotein cholesterol (non-HDL-C) and coronary artery disease (CAD). Two signals are novel with respect to association with non-HDL-C and are represented by non-coding low frequency variants (between 2–4% frequency), the splice region variant rs72658867-A in intron 14 and rs17248748-T in intron one. These two novel associations were replicated in three additional populations. Both variants lower non-HDL-C levels (rs72658867-A, non-HDL-C effect = -0.44 mmol/l, P adj = 1.1 × 10−80 and rs17248748-T, non-HDL-C effect = -0.13 mmol/l, P adj = 1.3 × 10−12) and confer protection against CAD (rs72658867-A, OR = 0.76 and P adj = 2.7 × 10−8 and rs17248748-T, OR = 0.92 and P adj = 0.022). The LDLR splice region variant, rs72658867-A, located at position +5 in intron 14 (NM_000527:c.2140+5G>A), causes retention of intron 14 during transcription and is expected to produce a truncated LDL receptor lacking domains essential for function of the receptor. About half of the transcripts generated from chromosomes carrying rs72658867-A are characterized by this retention of the intron. The same variant also increases LDLR mRNA expression, however, the wild type transcripts do not exceed levels in non-carriers. This demonstrates that sequence variants that disrupt the LDL receptor can lower non-HDL-C and protect against CAD.

A Missense Variant in PLEC Increases Risk of Atrial Fibrillation.

BACKGROUNDnGenome-wide association studies (GWAS) have yielded variants at >30 loci that associate with atrialxa0fibrillation (AF), including rare coding mutations in the sarcomere genes MYH6 and MYL4.nnnOBJECTIVESnThe aim of this study was to search for novel AF associations and in doing so gain insights into the mechanisms whereby variants affect AF risk, using electrocardiogram (ECG) measurements.nnnMETHODSnThe authors performed a GWAS of 14,255 AF cases and 374,939 controls, using whole-genome sequence data from the Icelandic population, and tested novel signals in 2,002 non-Icelandic cases and 12,324 controls. They then tested the AF variants for effect on cardiac electrical function by using measurements in 289,297 ECGs from 62,974 individuals.nnnRESULTSnThe authors discovered 2 novel AF variants, the intergenic variant rs72700114, between the genes LINC01142 and METTL11B (risk allele frequencyxa0=xa08.1%; odds ratio [OR]: 1.26; pxa0=xa03.1xa0× 10-18), and the missense variant p.Gly4098Ser in PLEC (frequencyxa0=xa01.2%; OR: 1.55; pxa0=xa08.0xa0× 10-10), encoding plectin, a cytoskeletal cross-linking protein that contributes to integrity of cardiac tissue. The authors also confirmed 29 reported variants. p.Gly4098Ser in PLEC significantly affects various ECG measurements in the absence of AF. Other AF variants have diverse effects on thexa0conduction system, ranging from none to extensive.nnnCONCLUSIONSnThe discovery of a missense variant in PLEC affecting AF combined with recent discoveries of variants inxa0the sarcomere genes MYH6 and MYL4 points to an important role of myocardial structure in the pathogenesis of thexa0disease. The diverse associations between AF variants and ECG measurements suggest fundamentally differentxa0categories of mechanisms contributing to the development of AF.

A rare splice donor mutation in the haptoglobin gene associates with blood lipid levels and coronary artery disease.

Common sequence variants at the haptoglobin gene (HP) have been associated with blood lipid levels. Through whole-genome sequencing of 8,453 Icelanders, we discovered a splice donor founder mutation in HP (NM_001126102.1:c.190u2009+u20091Gu2009>u2009C, minor allele frequencyu2009=u20090.56%). This mutation occurs on the HP1 allele of the common copy number variant in HP and leads to a loss of function of HP1. It associates with lower levels of haptoglobin (Pu2009=u20092.1u2009×u200910-54), higher levels of non-high density lipoprotein cholesterol (βu2009=u20090.26u2009mmol/l, Pu2009=u20092.6u2009×u200910-9) and greater risk of coronary artery disease (odds ratiou2009=u20091.30, 95% confidence interval: 1.10-1.54, Pu2009=u20090.0024). Through haplotype analysis and with RNA sequencing, we provide evidence of a causal relationship between one of the two haptoglobin isoforms, namely Hp1, and lower levels of non-HDL cholesterol. Furthermore, we show that the HP1 allele associates with various other quantitative biological traits.

Regulatory variants at KLF14 influence type 2 diabetes risk via a female-specific effect on adipocyte size and body composition

Individual risk of type 2 diabetes (T2D) is modified by perturbations to the mass, distribution and function of adipose tissue. To investigate the mechanismsxa0underlying these associations, we explored the molecular, cellular and whole-body effects of T2D-associated alleles near KLF14. We show that KLF14 diabetes-risk alleles act in adipose tissue to reduce KLF14 expression and modulate, in trans, the expression of 385 genes. We demonstrate, in human cellular studies, that reduced KLF14 expression increases pre-adipocyte proliferation but disrupts lipogenesis, and in mice, that adipose tissue–specific deletion of Klf14 partially recapitulates the human phenotype of insulin resistance, dyslipidemia and T2D. We show that carriers of the KLF14 T2D risk allele shift body fat from gynoid stores to abdominal stores and display a marked increase in adipocyte cell size, and that these effects on fat distribution, and the T2D association, are female specific. The metabolic risk associated with variation at this imprinted locus depends on the sex both of the subject and of the parent from whom the risk allele derives.Analysis of the imprinted KLF14 locus shows that the type 2 diabetes risk alleles in this region act in adipocytes to reduce KLF14 expression and modulate the expression of almost 400 genes in trans, leading to a shift in body-fat distribution and insulin resistance specifically in females.