Jette Bork-Jensen

Novel variation and de novo mutation rates in population-wide de novo assembled Danish trios

Building a population-specific catalogue of single nucleotide variants (SNVs), indels and structural variants (SVs) with frequencies, termed a national pan-genome, is critical for further advancing clinical and public health genetics in large cohorts. Here we report a Danish pan-genome obtained from sequencing 10 trios to high depth (50 × ). We report 536k novel SNVs and 283k novel short indels from mapping approaches and develop a population-wide de novo assembly approach to identify 132k novel indels larger than 10 nucleotides with low false discovery rates. We identify a higher proportion of indels and SVs than previous efforts showing the merits of high coverage and de novo assembly approaches. In addition, we use trio information to identify de novo mutations and use a probabilistic method to provide direct estimates of 1.27e−8 and 1.5e−9 per nucleotide per generation for SNVs and indels, respectively.

MicroRNA expression profiling of carcinoma in situ cells of the testis

Testicular germ cell tumours, seminoma (SE) and non-seminoma (NS), of young adult men develop from a precursor cell, carcinoma in situ (CIS), which resembles foetal gonocytes and retains embryonic pluripotency. We used microarrays to analyse microRNA (miRNA) expression in 12 human testis samples with CIS cells and compared it with miRNA expression profiles of normal adult testis, testis with Sertoli-cell-only that lacks germ cells, testis tumours (SE and embryonal carcinoma (EC), an undifferentiated component of NS) and foetal male and female gonads. Principal components analysis revealed distinct miRNA expression profiles characteristic for each of the different tissue types. We identified several miRNAs that were unique to testis with CIS cells, foetal gonads and testis tumours. These included miRNAs from the hsa-miR-371-373 and -302-367 clusters that have previously been reported in germ cell tumours and three miRNAs (hsa-miR-96, -141 and -200c) that were also expressed in human epididymis. We found several miRNAs that were upregulated in testis tumours: hsa-miR-9, -105 and -182-183-96 clusters were highly expressed in SE, while the hsa-miR-515-526 cluster was high in EC. We conclude that the miRNA expression profile changes during testis development and that the miRNA profile of adult testis with CIS cells shares characteristic similarities with the expression in foetal gonocytes.

Identification and Functional Characterization of G6PC2 Coding Variants Influencing Glycemic Traits Define an Effector Transcript at the G6PC2-ABCB11 Locus

Genome wide association studies (GWAS) for fasting glucose (FG) and insulin (FI) have identified common variant signals which explain 4.8% and 1.2% of trait variance, respectively. It is hypothesized that low-frequency and rare variants could contribute substantially to unexplained genetic variance. To test this, we analyzed exome-array data from up to 33,231 non-diabetic individuals of European ancestry. We found exome-wide significant (P<5×10-7) evidence for two loci not previously highlighted by common variant GWAS: GLP1R (p.Ala316Thr, minor allele frequency (MAF)=1.5%) influencing FG levels, and URB2 (p.Glu594Val, MAF = 0.1%) influencing FI levels. Coding variant associations can highlight potential effector genes at (non-coding) GWAS signals. At the G6PC2/ABCB11 locus, we identified multiple coding variants in G6PC2 (p.Val219Leu, p.His177Tyr, and p.Tyr207Ser) influencing FG levels, conditionally independent of each other and the non-coding GWAS signal. In vitro assays demonstrate that these associated coding alleles result in reduced protein abundance via proteasomal degradation, establishing G6PC2 as an effector gene at this locus. Reconciliation of single-variant associations and functional effects was only possible when haplotype phase was considered. In contrast to earlier reports suggesting that, paradoxically, glucose-raising alleles at this locus are protective against type 2 diabetes (T2D), the p.Val219Leu G6PC2 variant displayed a modest but directionally consistent association with T2D risk. Coding variant associations for glycemic traits in GWAS signals highlight PCSK1, RREB1, and ZHX3 as likely effector transcripts. These coding variant association signals do not have a major impact on the trait variance explained, but they do provide valuable biological insights.

Sequencing and de novo assembly of 150 genomes from Denmark as a population reference

Hundreds of thousands of human genomes are now being sequenced to characterize genetic variation and use this information to augment association mapping studies of complex disorders and other phenotypic traits. Genetic variation is identified mainly by mapping short reads to the reference genome or by performing local assembly. However, these approaches are biased against discovery of structural variants and variation in the more complex parts of the genome. Hence, large-scale de novo assembly is needed. Here we show that it is possible to construct excellent de novo assemblies from high-coverage sequencing with mate-pair libraries extending up to 20 kilobases. We report de novo assemblies of 150 individuals (50 trios) from the GenomeDenmark project. The quality of these assemblies is similar to those obtained using the more expensive long-read technology. We use the assemblies to identify a rich set of structural variants including many novel insertions and demonstrate how this variant catalogue enables further deciphering of known association mapping signals. We leverage the assemblies to provide 100 completely resolved major histocompatibility complex haplotypes and to resolve major parts of the Y chromosome. Our study provides a regional reference genome that we expect will improve the power of future association mapping studies and hence pave the way for precision medicine initiatives, which now are being launched in many countries including Denmark.

Human biallelic MFN2 mutations induce mitochondrial dysfunction, upper body adipose hyperplasia, and suppression of leptin expression

MFN2 encodes mitofusin 2, a membrane-bound mediator of mitochondrial membrane fusion and inter-organelle communication. MFN2 mutations cause axonal neuropathy, with associated lipodystrophy only occasionally noted, however homozygosity for the p.Arg707Trp mutation was recently associated with upper body adipose overgrowth. We describe similar massive adipose overgrowth with suppressed leptin expression in four further patients with biallelic MFN2 mutations and at least one p.Arg707Trp allele. Overgrown tissue was composed of normal-sized, UCP1-negative unilocular adipocytes, with mitochondrial network fragmentation, disorganised cristae, and increased autophagosomes. There was strong transcriptional evidence of mitochondrial stress signalling, increased protein synthesis, and suppression of signatures of cell death in affected tissue, whereas mitochondrial morphology and gene expression were normal in skin fibroblasts. These findings suggest that specific MFN2 mutations cause tissue-selective mitochondrial dysfunction with increased adipocyte proliferation and survival, confirm a novel form of excess adiposity with paradoxical suppression of leptin expression, and suggest potential targeted therapies. DOI: http://dx.doi.org/10.7554/eLife.23813.001

ExomeChip-Wide Analysis of 95 626 Individuals Identifies 10 Novel Loci Associated With QT and JT Intervals

Background: QT interval, measured through a standard ECG, captures the time it takes for the cardiac ventricles to depolarize and repolarize. JT interval is the component of the QT interval that reflects ventricular repolarization alone. Prolonged QT interval has been linked to higher risk of sudden cardiac arrest. Methods and Results: We performed an ExomeChip-wide analysis for both QT and JT intervals, including 209 449 variants, both common and rare, in 17 341 genes from the Illumina Infinium HumanExome BeadChip. We identified 10 loci that modulate QT and JT interval duration that have not been previously reported in the literature using single-variant statistical models in a meta-analysis of 95 626 individuals from 23 cohorts (comprised 83 884 European ancestry individuals, 9610 blacks, 1382 Hispanics, and 750 Asians). This brings the total number of ventricular repolarization associated loci to 45. In addition, our approach of using coding variants has highlighted the role of 17 specific genes for involvement in ventricular repolarization, 7 of which are in novel loci. Conclusions: Our analyses show a role for myocyte internal structure and interconnections in modulating QT interval duration, adding to previous known roles of potassium, sodium, and calcium ion regulation, as well as autonomic control. We anticipate that these discoveries will open new paths to the goal of making novel remedies for the prevention of lethal ventricular arrhythmias and sudden cardiac arrest.

Carriers of a VEGFA enhancer polymorphism selectively binding chop/ddit3 are predisposed to increased circulating levels of thyroid stimulating hormone

Background Levels of serum thyroid-stimulating hormone (TSH) indicate thyroid function, because thyroid hormone negatively controls TSH release. Genetic variants in the vascular endothelial growth factor A (VEGFA) gene are associated with TSH levels. The aim of this study was to characterise the association of VEGFA variants with TSH in a Danish cohort and to identify and characterise functional variants. Methods We performed an association study of the VEGFA locus for circulating TSH levels in 8445 Danish individuals. Lead variants were tested for allele-specific effects in vitro using luciferase reporter and gel-shift assays. Results Four SNPs in VEGFA were associated with circulating TSH (rs9472138, rs881858, rs943080 and rs4711751). For rs881858, the presence of each G-allele was associated with a corresponding decrease in TSH levels of 2.3% (p=8.4×10−9) and an increase in circulating free T4 levels (p=0.0014). The SNP rs881858 is located in a binding site for CHOP (C/EBP homology protein) and c/EBPβ (ccaat enhancer binding protein β). Reporter-gene analysis showed increased basal enhancer activity of the rs881858 A-allele versus the G-allele (34.5±9.9% (average±SEM), p=0.0012), while co-expression of CHOP effectively suppressed the rs881858 A-allele activity. The A-allele showed stronger binding to CHOP in gel-shift assays. Conclusions VEGF is an important angiogenic signal required for tissue expansion. We show that VEGFA variation giving allele-specific response to transcription factors with overlapping binding sites associate closely with circulating TSH levels. Because CHOP is induced by several types of intracellular stress, this indicates that cellular stress could be involved in the normal or pathophysiological response of the thyroid to TSH. Trial registration number NCT00289237, NCT00316667; Results.

Assembly and analysis of 100 full MHC haplotypes from the Danish population

Genes in the major histocompatibility complex (MHC, also known as HLA) play a critical role in the immune response and variation within the extended 4-Mb region shows association with major risks of many diseases. Yet, deciphering the underlying causes of these associations is difficult because the MHC is the most polymorphic region of the genome with a complex linkage disequilibrium structure. Here, we reconstruct full MHC haplotypes from de novo assembled trios without relying on a reference genome and perform evolutionary analyses. We report 100 full MHC haplotypes and call a large set of structural variants in the regions for future use in imputation with GWAS data. We also present the first complete analysis of the recombination landscape in the entire region and show how balancing selection at classical genes have linked effects on the frequency of variants throughout the region.

Exome-wide association study on Albuminuria identifies a novel rare variant in CUBN and additional genes, in 33,985 Europeans with and without diabetes

Identifying rare coding variants associated with albuminuria may open new avenues for preventing chronic kidney disease (CKD) and end-stage renal disease which are highly prevalent in patients with diabetes. Efforts to identify genetic susceptibility variants for albuminuria have so far been limited with the majority of studies focusing on common variants. We performed an exome-wide association study to identify coding variants in a two phase (discovery and replication) approach, totaling to 33,985 individuals of European ancestry (15,872 with and 18,113 without diabetes) and further testing in Greenlanders (n = 2,605). We identify a rare (MAF: 0.8%) missense (A1690V) variant in CUBN (rs141640975, β=0.27, p=1.3 × 10−11) associated with albuminuria as a continuous measure in the combined European meta-analyses. Presence of each rare allele of the variant was associated with a 6.4% increase in albuminuria. The rare CUBN variant had 3 times stronger effect in individuals with diabetes compared to those without (pinteraction: 5.4 × 10−4, βDM: 0.69, βnonDM: 0.20) in the discovery meta-analyses. Geneaggregate tests based on rare and common variants identify three additional genes associated with albuminuria (HES1, CDC73, and GRM5) after multiple testing correction (P_bonferroni<2.7 × 10−6). The current study identifies a rare coding variant in the CUBN locus and other potential genes associated with albuminuria in individuals with and without diabetes. These genes have been implicated in renal and cardiovascular dysfunction. These findings provide new insights into the genetic architecture of albuminuria and highlight novel target genes and pathways for prevention of diabetes-related kidney disease. Significance statement Increased albuminuria is a key manifestation of major health burdens, including chronic kidney disease and/or cardiovascular disease. Although being partially heritable, there is a lack of knowledge on rare genetic variants that contribute to albuminuria. The current study describes the discovery and validation, of a new rare gene mutation (~1%) in the CUBN gene which associates with increased albuminuria. Its effect multiplies 3 folds among diabetes cases compared to non diabetic individuals. The study further uncovers 3 additional genes modulating albuminuria levels in humans. Thus the current study findings provide new insights into the genetic architecture of albuminuria and highlight novel genes/pathways for prevention of diabetes related kidney disease.