Large-scale plasma proteomics comparisons through genetics and disease associations.

High-throughput proteomics platforms measuring thousands of proteins in plasma combined with genomic and phenotypic information have the power to bridge the gap between the genome and diseases. Here we performed association studies of Olink Explore 3072 data generated by the UK Biobank Pharma Proteomics Project1 on plasma samples from more than 50,000 UK Biobank participants with phenotypic and genotypic data, stratifying on British or Irish, African and South Asian ancestries. We compared the results with those of a SomaScan v4 study on plasma from 36,000 Icelandic people2, for 1,514 of whom Olink data were also available. We found modest correlation between the two platforms. Although cis protein quantitative trait loci were detected for a similar absolute number of assays on the two platforms (2,101 on Olink versus 2,120 on SomaScan), the proportion of assays with such supporting evidence for assay performance was higher on the Olink platform (72% versus 43%). A considerable number of proteins had genomic associations that differed between the platforms. We provide examples where differences between platforms may influence conclusions drawn from the integration of protein levels with the study of diseases. We demonstrate how leveraging the diverse ancestries of participants in the UK Biobank helps to detect novel associations and refine genomic location. Our results show the value of the information provided by the two most commonly used high-throughput proteomics platforms and demonstrate the differences between them that at times provides useful complementarity.

The sequences of 150,119 genomes in the UK Biobank.

Detailed knowledge of how diversity in the sequence of the human genome affects phenotypic diversity depends on a comprehensive and reliable characterization of both sequences and phenotypic variation. Over the past decade, insights into this relationship have been obtained from whole-exome sequencing or whole-genome sequencing of large cohorts with rich phenotypic data1,2. Here we describe the analysis of whole-genome sequencing of 150,119 individuals from the UK Biobank3. This constitutes a set of high-quality variants, including 585,040,410 single-nucleotide polymorphisms, representing 7.0% of all possible human single-nucleotide polymorphisms, and 58,707,036 indels. This large set of variants allows us to characterize selection based on sequence variation within a population through a depletion rank score of windows along the genome. Depletion rank analysis shows that coding exons represent a small fraction of regions in the genome subject to strong sequence conservation. We define three cohorts within the UK Biobank: a large British Irish cohort, a smaller African cohort and a South Asian cohort. A haplotype reference panel is provided that allows reliable imputation of most variants carried by three or more sequenced individuals. We identified 895,055 structural variants and 2,536,688 microsatellites, groups of variants typically excluded from large-scale whole-genome sequencing studies. Using this formidable new resource, we provide several examples of trait associations for rare variants with large effects not found previously through studies based on whole-exome sequencing and/or imputation.

Actionable Genotypes and Their Association with Life Span in Iceland.

BACKGROUND: In 2021, the American College of Medical Genetics and Genomics (ACMG) recommended reporting actionable genotypes in 73 genes associated with diseases for which preventive or therapeutic measures are available. Evaluations of the association of actionable genotypes in these genes with life span are currently lacking. METHODS: We assessed the prevalence of coding and splice variants in genes on the ACMG Secondary Findings, version 3.0 (ACMG SF v3.0), list in the genomes of 57,933 Icelanders. We assigned pathogenicity to all reviewed variants using reported evidence in the ClinVar database, the frequency of variants, and their associations with disease to create a manually curated set of actionable genotypes (variants). We assessed the relationship between these genotypes and life span and further examined the specific causes of death among carriers. RESULTS: Through manual curation of 4405 sequence variants in the ACMG SF v3.0 genes, we identified 235 actionable genotypes in 53 genes. Of the 57,933 participants, 2306 (4.0%) carried at least one actionable genotype. We found shorter median survival among persons carrying actionable genotypes than among noncarriers. Specifically, we found that carrying an actionable genotype in a cancer gene was associated with survival that was 3 years shorter than that among noncarriers, with causes of death among carriers attributed primarily to cancer-related conditions. Furthermore, we found evidence of association between carrying an actionable genotype in certain genes in the cardiovascular disease group and a reduced life span. CONCLUSIONS: On the basis of the ACMG SF v3.0 guidelines, we found that approximately 1 in 25 Icelanders carried an actionable genotype and that carrying such a genotype was associated with a reduced life span. (Funded by deCODE Genetics-Amgen.).

Multiomics analysis of rheumatoid arthritis yields sequence variants that have large effects on risk of the seropositive subset.

OBJECTIVES: To find causal genes for rheumatoid arthritis (RA) and its seropositive (RF and/or ACPA positive) and seronegative subsets. METHODS: We performed a genome-wide association study (GWAS) of 31 313 RA cases (68% seropositive) and ~1 million controls from Northwestern Europe. We searched for causal genes outside the HLA-locus through effect on coding, mRNA expression in several tissues and/or levels of plasma proteins (SomaScan) and did network analysis (Qiagen). RESULTS: We found 25 sequence variants for RA overall, 33 for seropositive and 2 for seronegative RA, altogether 37 sequence variants at 34 non-HLA loci, of which 15 are novel. Genomic, transcriptomic and proteomic analysis of these yielded 25 causal genes in seropositive RA and additional two overall. Most encode proteins in the network of interferon-alpha/beta and IL-12/23 that signal through the JAK/STAT-pathway. Highlighting those with largest effect on seropositive RA, a rare missense variant in STAT4 (rs140675301-A) that is independent of reported non-coding STAT4-variants, increases the risk of seropositive RA 2.27-fold (p=2.1×10-9), more than the rs2476601-A missense variant in PTPN22 (OR=1.59, p=1.3×10-160). STAT4 rs140675301-A replaces hydrophilic glutamic acid with hydrophobic valine (Glu128Val) in a conserved, surface-exposed loop. A stop-mutation (rs76428106-C) in FLT3 increases seropositive RA risk (OR=1.35, p=6.6×10-11). Independent missense variants in TYK2 (rs34536443-C, rs12720356-C, rs35018800-A, latter two novel) associate with decreased risk of seropositive RA (ORs=0.63-0.87, p=10-9-10-27) and decreased plasma levels of interferon-alpha/beta receptor 1 that signals through TYK2/JAK1/STAT4. CONCLUSION: Sequence variants pointing to causal genes in the JAK/STAT pathway have largest effect on seropositive RA, while associations with seronegative RA remain scarce.

Clonal hematopoiesis, with and without candidate driver mutations, is common in the elderly.

Clonal hematopoiesis (CH) arises when a substantial proportion of mature blood cells is derived from a single dominant hematopoietic stem cell lineage. Somatic mutations in candidate driver (CD) genes are thought to be responsible for at least some cases of CH. Using whole-genome sequencing of 11 262 Icelanders, we found 1403 cases of CH by using barcodes of mosaic somatic mutations in peripheral blood, whether or not they have a mutation in a CD gene. We find that CH is very common in the elderly, trending toward inevitability. We show that somatic mutations in TET2, DNMT3A, ASXL1, and PPM1D are associated with CH at high significance. However, known CD mutations were evident in only a fraction of CH cases. Nevertheless, the highly prevalent CH we detect associates with increased mortality rates, risk for hematological malignancy, smoking behavior, telomere length, Y-chromosome loss, and other phenotypic characteristics. Modeling suggests some CH cases could arise in the absence of CD mutations as a result of neutral drift acting on a small population of active hematopoietic stem cells. Finally, we find a germline deletion in intron 3 of the telomerase reverse transcriptase (TERT) gene that predisposes to CH (rs34002450; P = 7.4 × 10-12; odds ratio, 1.37).

Insertion of an SVA-E retrotransposon into the CASP8 gene is associated with protection against prostate cancer.

Transcriptional and splicing anomalies have been observed in intron 8 of the CASP8 gene (encoding procaspase-8) in association with cutaneous basal-cell carcinoma (BCC) and linked to a germline SNP rs700635. Here, we show that the rs700635[C] allele, which is associated with increased risk of BCC and breast cancer, is protective against prostate cancer [odds ratio (OR) = 0.91, P = 1.0 × 10(-6)]. rs700635[C] is also associated with failures to correctly splice out CASP8 intron 8 in breast and prostate tumours and in corresponding normal tissues. Investigation of rs700635[C] carriers revealed that they have a human-specific short interspersed element-variable number of tandem repeat-Alu (SINE-VNTR-Alu), subfamily-E retrotransposon (SVA-E) inserted into CASP8 intron 8. The SVA-E shows evidence of prior activity, because it has transduced some CASP8 sequences during subsequent retrotransposition events. Whole-genome sequence (WGS) data were used to tag the SVA-E with a surrogate SNP rs1035142[T] (r(2) = 0.999), which showed associations with both the splicing anomalies (P = 6.5 × 10(-32)) and with protection against prostate cancer (OR = 0.91, P = 3.8 × 10(-7)).

Rate of de novo mutations and the importance of father's age to disease risk.

Mutations generate sequence diversity and provide a substrate for selection. The rate of de novo mutations is therefore of major importance to evolution. Here we conduct a study of genome-wide mutation rates by sequencing the entire genomes of 78 Icelandic parent-offspring trios at high coverage. We show that in our samples, with an average father's age of 29.7, the average de novo mutation rate is 1.20 × 10(-8) per nucleotide per generation. Most notably, the diversity in mutation rate of single nucleotide polymorphisms is dominated by the age of the father at conception of the child. The effect is an increase of about two mutations per year. An exponential model estimates paternal mutations doubling every 16.5 years. After accounting for random Poisson variation, father's age is estimated to explain nearly all of the remaining variation in the de novo mutation counts. These observations shed light on the importance of the father's age on the risk of diseases such as schizophrenia and autism.

A frameshift deletion in the sarcomere gene MYL4 causes early-onset familial atrial fibrillation.

AIMS: Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia in man, causing substantial morbidity and mortality with a major worldwide public health impact. It is increasingly recognized as a highly heritable condition. This study aimed to determine genetic risk factors for early-onset AF. METHODS AND RESULTS: We sequenced the whole genomes of 8453 Icelanders and imputed genotypes of the 25.5 million sequence variants we discovered into 1799 Icelanders with early-onset AF (diagnosed before 60 years of age) and 337 453 controls. Each sequence variant was tested for association based on multiplicative and recessive inheritance models. We discovered a rare frameshift deletion in the myosin MYL4 gene (c.234delC) that associates with early-onset AF under a recessive mode of inheritance (allelic frequency = 0.58%). We found eight homozygous carriers of the mutation, all of whom had early-onset AF. Six of the homozygotes were diagnosed by the age of 30 and the remaining two in their 50s. Three of the homozygotes had received pacemaker implantations due to sick sinus syndrome, three had suffered an ischemic stroke, and one suffered sudden cardiac death. CONCLUSIONS: Through a population approach we found a loss of function mutation in the myosin gene MYL4 that, in the homozygous state, is completely penetrant for early-onset AF. The finding may provide novel mechanistic insight into the pathophysiology of this complex arrhythmia.

GORpipe: a query tool for working with sequence data based on a Genomic Ordered Relational (GOR) architecture.

MOTIVATION: Our aim was to create a general-purpose relational data format and analysis tools to provide an efficient and coherent framework for working with large volumes of DNA sequence data. RESULTS: For this purpose we developed the GORpipe software system. It is based on a genomic ordered architecture and uses a declarative query language that combines features from SQL and shell pipe syntax in a novel manner. The system can for instance be used to annotate sequence variants, find genomic spatial overlap between various types of genomic features, filter and aggregate them in various ways. AVAILABILITY AND IMPLEMENTATION: The GORpipe software is freely available for non-commercial academic usage and can be downloaded from www.nextcode.com/gorpipe CONTACT: hakon@wuxinextcode.comSupplementary information: Supplementary data are available at Bioinformatics online.

Compound heterozygous mutations in UBA5 causing early-onset epileptic encephalopathy in two sisters.

BACKGROUND: Epileptic encephalopathies are a group of childhood epilepsies that display high phenotypic and genetic heterogeneity. The recent, extensive use of next-generation sequencing has identified a large number of genes in epileptic encephalopathies, including UBA5 in which biallelic mutations were first described as pathogenic in 2016 (Colin E et al., Am J Hum Genet 99(3):695-703, 2016. Muona M et al., Am J Hum Genet 99(3):683-694, 2016). UBA5 encodes an activating enzyme for a post-translational modification mechanism known as ufmylation, and is the first gene from the ufmylation pathway that is linked to disease. CASE PRESENTATION: We sequenced the genomes of two sisters with early-onset epileptic encephalopathy along with their unaffected parents in an attempt to find a genetic cause for their condition. The sisters, born in 2004 and 2006, presented with infantile spasms at six months of age, which later progressed to recurrent, treatment-resistant seizures. We detected a compound heterozygous genotype in UBA5 in the sisters, a genotype not seen elsewhere in an Icelandic reference set of 30,067 individuals nor in public databases. One of the mutations, c.684G > A, is a paternally inherited exonic splicing mutation, occuring at the last nucleotide of exon 7 of UBA5. The mutation is predicted to disrupt the splice site, resulting in loss-of-function of one allele of UBA5. The second mutation is a maternally inherited missense mutation, p.Ala371Thr, previously reported as pathogenic when in compound heterozygosity with a loss-of-function mutation in UBA5 and is believed to produce a hypomorphic allele. Supportive of this, we have identified three adult Icelanders homozygous for the p.Ala371Thr mutation who show no signs of neurological disease. CONCLUSIONS: We describe compound heterozygous mutations in the UBA5 gene in two sisters with early-onset epileptic encephalopathy. To our knowledge, this is the first description of mutations in UBA5 since the initial discovery that pathogenic biallelic variants in the gene cause early-onset epileptic encephalopathy. We further provide confirmatory evidence that p.Ala371Thr is a hypomorphic mutation, by presenting three adult homozygotes who show no signs of neurological disease.

Germline sequence variants in TGM3 and RGS22 confer risk of basal cell carcinoma.

To search for new sequence variants that confer risk of cutaneous basal cell carcinoma (BCC), we conducted a genome-wide association study of 38.5 million single nucleotide polymorphisms (SNPs) and small indels identified through whole-genome sequencing of 2230 Icelanders. We imputed genotypes for 4208 BCC patients and 109 408 controls using Illumina SNP chip typing data, carried out association tests and replicated the findings in independent population samples. We found new BCC susceptibility loci at TGM3 (rs214782[G], P = 5.5 × 10(-17), OR = 1.29) and RGS22 (rs7006527[C], P = 8.7 × 10(-13), OR = 0.77). TGM3 encodes transglutaminase type 3, which plays a key role in production of the cornified envelope during epidermal differentiation.

Genome-wide association study yields variants at 20p12.2 that associate with urinary bladder cancer.

Genome-wide association studies (GWAS) of urinary bladder cancer (UBC) have yielded common variants at 12 loci that associate with risk of the disease. We report here the results of a GWAS of UBC including 1670 UBC cases and 90 180 controls, followed by replication analysis in additional 5266 UBC cases and 10 456 controls. We tested a dataset containing 34.2 million variants, generated by imputation based on whole-genome sequencing of 2230 Icelanders. Several correlated variants at 20p12, represented by rs62185668, show genome-wide significant association with UBC after combining discovery and replication results (OR = 1.19, P = 1.5 × 10(-11) for rs62185668-A, minor allele frequency = 23.6%). The variants are located in a non-coding region approximately 300 kb upstream from the JAG1 gene, an important component of the Notch signaling pathways that may be oncogenic or tumor suppressive in several forms of cancer. Our results add to the growing number of UBC risk variants discovered through GWAS.

Fine-scale recombination rate differences between sexes, populations and individuals.

Meiotic recombinations contribute to genetic diversity by yielding new combinations of alleles. Recently, high-resolution recombination maps were inferred from high-density single-nucleotide polymorphism (SNP) data using linkage disequilibrium (LD) patterns that capture historical recombination events. The use of these maps has been demonstrated by the identification of recombination hotspots and associated motifs, and the discovery that the PRDM9 gene affects the proportion of recombinations occurring at hotspots. However, these maps provide no information about individual or sex differences. Moreover, locus-specific demographic factors like natural selection can bias LD-based estimates of recombination rate. Existing genetic maps based on family data avoid these shortcomings, but their resolution is limited by relatively few meioses and a low density of markers. Here we used genome-wide SNP data from 15,257 parent-offspring pairs to construct the first recombination maps based on directly observed recombinations with a resolution that is effective down to 10 kilobases (kb). Comparing male and female maps reveals that about 15% of hotspots in one sex are specific to that sex. Although male recombinations result in more shuffling of exons within genes, female recombinations generate more new combinations of nearby genes. We discover novel associations between recombination characteristics of individuals and variants in the PRDM9 gene and we identify new recombination hotspots. Comparisons of our maps with two LD-based maps inferred from data of HapMap populations of Utah residents with ancestry from northern and western Europe (CEU) and Yoruba in Ibadan, Nigeria (YRI) reveal population differences previously masked by noise and map differences at regions previously described as targets of natural selection.

Common variants on chromosomes 2q35 and 16q12 confer susceptibility to estrogen receptor-positive breast cancer.

Familial clustering studies indicate that breast cancer risk has a substantial genetic component. To identify new breast cancer risk variants, we genotyped approximately 300,000 SNPs in 1,600 Icelandic individuals with breast cancer and 11,563 controls using the Illumina Hap300 platform. We then tested selected SNPs in five replication sample sets. Overall, we studied 4,554 affected individuals and 17,577 controls. Two SNPs consistently associated with breast cancer: approximately 25% of individuals of European descent are homozygous for allele A of rs13387042 on chromosome 2q35 and have an estimated 1.44-fold greater risk than noncarriers, and for allele T of rs3803662 on 16q12, about 7% are homozygous and have a 1.64-fold greater risk. Risk from both alleles was confined to estrogen receptor-positive tumors. At present, no genes have been identified in the linkage disequilibrium block containing rs13387042. rs3803662 is near the 5' end of TNRC9 , a high mobility group chromatin-associated protein whose expression is implicated in breast cancer metastasis to bone.

Parental origin of sequence variants associated with complex diseases.

Effects of susceptibility variants may depend on from which parent they are inherited. Although many associations between sequence variants and human traits have been discovered through genome-wide associations, the impact of parental origin has largely been ignored. Here we show that for 38,167 Icelanders genotyped using single nucleotide polymorphism (SNP) chips, the parental origin of most alleles can be determined. For this we used a combination of genealogy and long-range phasing. We then focused on SNPs that associate with diseases and are within 500 kilobases of known imprinted genes. Seven independent SNP associations were examined. Five-one with breast cancer, one with basal-cell carcinoma and three with type 2 diabetes-have parental-origin-specific associations. These variants are located in two genomic regions, 11p15 and 7q32, each harbouring a cluster of imprinted genes. Furthermore, we observed a novel association between the SNP rs2334499 at 11p15 and type 2 diabetes. Here the allele that confers risk when paternally inherited is protective when maternally transmitted. We identified a differentially methylated CTCF-binding site at 11p15 and demonstrated correlation of rs2334499 with decreased methylation of that site.

Start codon variant in LAG3 is associated with decreased LAG-3 expression and increased risk of autoimmune thyroid disease.

Autoimmune thyroid disease (AITD) is a common autoimmune disease. In a GWAS meta-analysis of 110,945 cases and 1,084,290 controls, 290 sequence variants at 225 loci are associated with AITD. Of these variants, 115 are previously unreported. Multiomics analysis yields 235 candidate genes outside the MHC-region and the findings highlight the importance of genes involved in T-cell regulation. A rare 5'-UTR variant (rs781745126-T, MAF = 0.13% in Iceland) in LAG3 has the largest effect (OR = 3.42, P = 2.2 × 10-16) and generates a novel start codon for an open reading frame upstream of the canonical protein translation initiation site. rs781745126-T reduces mRNA and surface expression of the inhibitory immune checkpoint LAG-3 co-receptor on activated lymphocyte subsets and halves LAG-3 levels in plasma among heterozygotes. All three homozygous carriers of rs781745126-T have AITD, of whom one also has two other T-cell mediated diseases, that is vitiligo and type 1 diabetes. rs781745126-T associates nominally with vitiligo (OR = 5.1, P = 6.5 × 10-3) but not with type 1 diabetes. Thus, the effect of rs781745126-T is akin to drugs that inhibit LAG-3, which unleash immune responses and can have thyroid dysfunction and vitiligo as adverse events. This illustrates how a multiomics approach can reveal potential drug targets and safety concerns.

Sequence variants at CYP1A1-CYP1A2 and AHR associate with coffee consumption.

Coffee is the most commonly used stimulant and caffeine is its main psychoactive ingredient. The heritability of coffee consumption has been estimated at around 50%. We performed a meta-analysis of four genome-wide association studies of coffee consumption among coffee drinkers from Iceland (n = 2680), The Netherlands (n = 2791), the Sorbs Slavonic population isolate in Germany (n = 771) and the USA (n = 369) using both directly genotyped and imputed single nucleotide polymorphisms (SNPs) (2.5 million SNPs). SNPs at the two most significant loci were also genotyped in a sample set from Iceland (n = 2430) and a Danish sample set consisting of pregnant women (n = 1620). Combining all data, two sequence variants significantly associated with increased coffee consumption: rs2472297-T located between CYP1A1 and CYP1A2 at 15q24 (P = 5.4 · 10(-14)) and rs6968865-T near aryl hydrocarbon receptor (AHR) at 7p21 (P = 2.3 · 10(-11)). An effect of ∼0.2 cups a day per allele was observed for both SNPs. CYP1A2 is the main caffeine metabolizing enzyme and is also involved in drug metabolism. AHR detects xenobiotics, such as polycyclic aryl hydrocarbons found in roasted coffee, and induces transcription of CYP1A1 and CYP1A2. The association of these SNPs with coffee consumption was present in both smokers and non-smokers.

Ancestry-shift refinement mapping of the C6orf97-ESR1 breast cancer susceptibility locus.

We used an approach that we term ancestry-shift refinement mapping to investigate an association, originally discovered in a GWAS of a Chinese population, between rs2046210[T] and breast cancer susceptibility. The locus is on 6q25.1 in proximity to the C6orf97 and estrogen receptor alpha (ESR1) genes. We identified a panel of SNPs that are correlated with rs2046210 in Chinese, but not necessarily so in other ancestral populations, and genotyped them in breast cancer case:control samples of Asian, European, and African origin, a total of 10,176 cases and 13,286 controls. We found that rs2046210[T] does not confer substantial risk of breast cancer in Europeans and Africans (OR = 1.04, P = 0.099, and OR = 0.98, P = 0.77, respectively). Rather, in those ancestries, an association signal arises from a group of less common SNPs typified by rs9397435. The rs9397435[G] allele was found to confer risk of breast cancer in European (OR = 1.15, P = 1.2 x 10(-3)), African (OR = 1.35, P = 0.014), and Asian (OR = 1.23, P = 2.9 x 10(-4)) population samples. Combined over all ancestries, the OR was 1.19 (P = 3.9 x 10(-7)), was without significant heterogeneity between ancestries (P(het) = 0.36) and the SNP fully accounted for the association signal in each ancestry. Haplotypes bearing rs9397435[G] are well tagged by rs2046210[T] only in Asians. The rs9397435[G] allele showed associations with both estrogen receptor positive and estrogen receptor negative breast cancer. Using early-draft data from the 1,000 Genomes project, we found that the risk allele of a novel SNP (rs77275268), which is closely correlated with rs9397435, disrupts a partially methylated CpG sequence within a known CTCF binding site. These studies demonstrate that shifting the analysis among ancestral populations can provide valuable resolution in association mapping.

A high-resolution recombination map of the human genome.

Determination of recombination rates across the human genome has been constrained by the limited resolution and accuracy of existing genetic maps and the draft genome sequence. We have genotyped 5,136 microsatellite markers for 146 families, with a total of 1,257 meiotic events, to build a high-resolution genetic map meant to: (i) improve the genetic order of polymorphic markers; (ii) improve the precision of estimates of genetic distances; (iii) correct portions of the sequence assembly and SNP map of the human genome; and (iv) build a map of recombination rates. Recombination rates are significantly correlated with both cytogenetic structures (staining intensity of G bands) and sequence (GC content, CpG motifs and poly(A)/poly(T) stretches). Maternal and paternal chromosomes show many differences in locations of recombination maxima. We detected systematic differences in recombination rates between mothers and between gametes from the same mother, suggesting that there is some underlying component determined by both genetic and environmental factors that affects maternal recombination rates.