Isleifur Olafsson

Variants in ELL2 influencing immunoglobulin levels associate with multiple myeloma

Multiple myeloma (MM) is characterized by an uninhibited, clonal growth of plasma cells. While first-degree relatives of patients with MM show an increased risk of MM, the genetic basis of inherited MM susceptibility is incompletely understood. Here we report a genome-wide association study in the Nordic region identifying a novel MM risk locus at ELL2 (rs56219066T; odds ratio (OR)=1.25; P=9.6 × 10−10). This gene encodes a stoichiometrically limiting component of the super-elongation complex that drives secretory-specific immunoglobulin mRNA production and transcriptional regulation in plasma cells. We find that the MM risk allele harbours a Thr298Ala missense variant in an ELL2 domain required for transcription elongation. Consistent with a hypomorphic effect, we find that the MM risk allele also associates with reduced levels of immunoglobulin A (IgA) and G (IgG) in healthy subjects (P=8.6 × 10−9 and P=6.4 × 10−3, respectively) and, potentially, with an increased risk of bacterial meningitis (OR=1.30; P=0.0024).

Large-scale whole-genome sequencing of the Icelandic population

Here we describe the insights gained from sequencing the whole genomes of 2,636 Icelanders to a median depth of 20×. We found 20 million SNPs and 1.5 million insertions-deletions (indels). We describe the density and frequency spectra of sequence variants in relation to their functional annotation, gene position, pathway and conservation score. We demonstrate an excess of homozygosity and rare protein-coding variants in Iceland. We imputed these variants into 104,220 individuals down to a minor allele frequency of 0.1% and found a recessive frameshift mutation in MYL4 that causes early-onset atrial fibrillation, several mutations in ABCB4 that increase risk of liver diseases and an intronic variant in GNAS associating with increased thyroid-stimulating hormone levels when maternally inherited. These data provide a study design that can be used to determine how variation in the sequence of the human genome gives rise to human diversity.

Molecular cloning and sequence analysis of cDNA coding for the precursor of the human cysteine proteinase inhibitor cystatin C

Recombinant cystatin C producing clones were isolated from a human placenta λgt11 cDNA library. The cDNA insert of one of the clones, containing 777 base pairs, encodes the complete mature cystatin C (120 amino acids) and a hydrophobic leader sequence of 26 amino acids, indicating an extracellular function of the inhibitor. The deduced protein sequence confirms the protein sequence of cystatin C isolated from human urine, but differs in one position from the sequence of the cystatin C fragment deposited as amyloid in hereditary cerebral hemorrhage with amyloidosis.

Mutation in cystatin C gene causes hereditary brain haemorrhage

Hereditary cystatin C amyloid angiopathy (HCCAA) is an autosomal dominant disorder in which a cysteine proteinase inhibitor, cystatin C, is deposited as amyloid fibrils in the cerebral arteries of patients and leads to massive brain haemorrhage and death in young adults. A full length cystatin C cDNA probe revealed a mutation in the codon for leucine at position 68 which abolishes an Alu I restriction site in the cystatin C gene of HCCAA patients. The Alu I marker has been used to show that this mutation is transmitted only in affected members of all eight families investigated, and that the mutated cystatin C gene causes HCCAA.

Nonsense mutation in the LGR4 gene is associated with several human diseases and other traits

Low bone mineral density (BMD) is used as a parameter of osteoporosis. Genome-wide association studies of BMD have hitherto focused on BMD as a quantitative trait, yielding common variants of small effects that contribute to the population diversity in BMD. Here we use BMD as a dichotomous trait, searching for variants that may have a direct effect on the risk of pathologically low BMD rather than on the regulation of BMD in the healthy population. Through whole-genome sequencing of Icelandic individuals, we found a rare nonsense mutation within the leucine-rich-repeat-containing G-protein-coupled receptor 4 (LGR4) gene (c.376C>T) that is strongly associated with low BMD, and with osteoporotic fractures. This mutation leads to termination of LGR4 at position 126 and fully disrupts its function. The c.376C>T mutation is also associated with electrolyte imbalance, late onset of menarche and reduced testosterone levels, as well as an increased risk of squamous cell carcinoma of the skin and biliary tract cancer. Interestingly, the phenotype of carriers of the c.376C>T mutation overlaps that of Lgr4 mutant mice.

Efficient production of native, biologically active human cystatin C by Escherichia coli

A cDNA encoding the mature human cysteine proteinase inhibitor cystatin C was fused to the coding sequence for the Escherichia coli outer membrane protein A signal peptide, and the recombinant gene was expressed in E. coli under the control of the λ PR promoter, an optimized Shine‐Dalgarno sequence and the λ cI 857 repressor. When induced at 42°C, such cells expressed large amounts of recombinant cystatin C. The recombinant protein was isolated in high yield and characterized. All physicochemical properties investigated, including the positions of disulfide bonds, indicated that the E. coli derived cystatin C was identical to cystatin C isolated from human biological fluids, except that the proline residue in position three was not hydroxylated. The recombinant protein displayed full biological activity against papain, cathepsin B and dipeptidyl peptidase I.

Promoter-mediated, dexamethasone-induced increase in cystatin C production by HeLa cells

Cystatin C, an efficient inhibitor of cysteine proteinases, is present in all investigated human extracellular fluids. Dexamethasone caused a significant and dose-dependent increase in the cystatin C secretion of cultivated HeLa cells up to a maximal increase of 80% at 10(-6) mol l-1 dexamethasone. Increased production of cystatin C was also observed at lower concentrations, suggesting that glucocorticoids might play a physiological role in the production of cystatin C. The effect of dexamethasone on the cystatin C gene expression was also studied in a transient transfection expression system using chimeric plasmid constructs of the cystatin C gene promoter (positions -2 to -1084) coupled to the structural gene for human growth hormone (hGH). In this system, a small, but statistically significant, increase in hGH secretion was also observed upon dexamethasone treatment, suggesting that the glucocorticoid-induced increase in secretion of cystatin C is due to a promoter-mediated increase in transcription of the cystatin C gene.

C reactive protein levels are increased in non-allergic but not allergic asthma: a multicentre epidemiological study

Background: High sensitivity C reactive protein (HsCRP) is an inflammatory marker known to be related to smoking, obesity, and cardiovascular disease. A study was undertaken to determine whether HsCRP is related to respiratory symptoms, asthma, atopy, and bronchial hyperresponsiveness in population samples from three countries. Methods: HsCRP was measured in 1289 subjects from three centres in ECRHS II: Reykjavik, Uppsala and Tartu. The HsCRP values ranged from <0.01 mg/l to 70.0 mg/l and were divided into four equal groups (⩽0.45, 0.46–0.96, 0.97–2.21, and >2.21 mg/l). Results: HsCRP increased with increasing body mass index (r = 0.41; p<0.0001) and was higher in smokers than in never smokers (p = 0.02). A significant relationship was found between increased HsCRP levels and respiratory symptoms such as wheeze, attacks of breathlessness after effort, and nocturnal cough (p<0.0001). The crude odds ratio (95% CI) for the probability of non-allergic asthma was 3.57 (1.83 to 6.96) for subjects in the 4th quartile compared with the 1st quartile of HsCRP. This association remained significant after adjusting for study centre, age, sex, body weight, and smoking history (OR 2.19 (95% CI 1.04 to 4.63)). No significant relationship was observed between HsCRP and allergic asthma or bronchial responsiveness. Conclusions: Raised levels of HsCRP are significantly associated with respiratory symptoms and non-allergic asthma but not with allergic asthma.

Discovery of common variants associated with low TSH levels and thyroid cancer risk

To search for sequence variants conferring risk of nonmedullary thyroid cancer, we focused our analysis on 22 SNPs with a P < 5 × 10−8 in a genome-wide association study on levels of thyroid stimulating hormone (TSH) in 27,758 Icelanders. Of those, rs965513 has previously been shown to associate with thyroid cancer. The remaining 21 SNPs were genotyped in 561 Icelandic individuals with thyroid cancer (cases) and up to 40,013 controls. Variants suggestively associated with thyroid cancer (P < 0.05) were genotyped in an additional 595 non-Icelandic cases and 2,604 controls. After combining the results, three variants were shown to associate with thyroid cancer: rs966423 on 2q35 (OR = 1.34; Pcombined = 1.3 × 10−9), rs2439302 on 8p12 (OR = 1.36; Pcombined = 2.0 × 10−9) and rs116909374 on 14q13.3 (OR = 2.09; Pcombined = 4.6 × 10−11), a region previously reported to contain an uncorrelated variant conferring risk of thyroid cancer. A strong association (P = 9.1 × 10−91) was observed between rs2439302 on 8p12 and expression of NRG1, which encodes the signaling protein neuregulin 1, in blood.

Genetic correction of PSA values using sequence variants associated with PSA levels.

Sequence variants in the human genome are associated with serum levels of prostate-specific antigen. SNPping Away at Prostate Cancer Measuring prostate-specific antigen (PSA) in serum is the only diagnostic test for prostate cancer and is used as a screening tool for deciding whether to perform a biopsy. Yet, this diagnostic test is far from ideal, with more than a third of men with serum PSA levels of 10 ng/ml or greater having no evidence of prostate cancer at biopsy, and some men with very low PSA levels (less than the lower threshold of 2.5 ng/ml), who are not given a biopsy but yet end up having prostate cancer. The lack of specificity and sensitivity of the PSA test and the many confounding factors that influence the test result, including medications, inflammation, and, of course, genotype, have reduced the value of this screening tool. As with most cancers, early detection of prostate cancer leads to a greatly improved chance of survival, so improving the predictive accuracy of this test is of paramount importance. In an effort to investigate whether genome sequence variants can be used to make the PSA test more sensitive, Gudmundsson and colleagues have undertaken a genome-wide association study in 15,757 Icelandic men and 454 British men not yet diagnosed with prostate cancer to see whether they can tie sequence variants [single-nucleotide polymorphisms (SNPs)] to serum PSA levels. The authors identify six loci with SNPs that correlate with PSA levels. They then probed these data more deeply. They looked at these loci in 3834 men who underwent subsequent biopsy of the prostate and demonstrate that three of these loci (10q26, 12q24, and 19q13.33) are associated not only with higher PSA levels but also with a higher probability of a negative biopsy result. The authors suggest that this genotype information should be used to calculate a personalized “cutoff” value for serum PSA levels in each individual to improve the predictive accuracy of the test and to ensure that only men who need a prostate biopsy are subjected to this procedure. Measuring serum levels of the prostate-specific antigen (PSA) is the most common screening method for prostate cancer. However, PSA levels are affected by a number of factors apart from neoplasia. Notably, around 40% of the variability of PSA levels in the general population is accounted for by inherited factors, suggesting that it may be possible to improve both sensitivity and specificity by adjusting test results for genetic effects. To search for sequence variants that associate with PSA levels, we performed a genome-wide association study and follow-up analysis using PSA information from 15,757 Icelandic and 454 British men not diagnosed with prostate cancer. Overall, we detected a genome-wide significant association between PSA levels and single-nucleotide polymorphisms (SNPs) at six loci: 5p15.33 (rs2736098), 10q11 (rs10993994), 10q26 (rs10788160), 12q24 (rs11067228), 17q12 (rs4430796), and 19q13.33 [rs17632542 (KLK3: I179T)], each with Pcombined <3 × 10−10. Among 3834 men who underwent a biopsy of the prostate, the 10q26, 12q24, and 19q13.33 alleles that associate with high PSA levels are associated with higher probability of a negative biopsy (odds ratio between 1.15 and 1.27). Assessment of association between the six loci and prostate cancer risk in 5325 cases and 41,417 controls from Iceland, the Netherlands, Spain, Romania, and the United States showed that the SNPs at 10q26 and 12q24 were exclusively associated with PSA levels, whereas the other four loci also were associated with prostate cancer risk. We propose that a personalized PSA cutoff value, based on genotype, should be used when deciding to perform a prostate biopsy.