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Marfan syndrome is an autosomal dominant condition characterized by aortic aneurysm, skeletal abnormalities, and lens dislocation, and is caused by mutations in the FBN1 gene. To explore causes of Marfan syndrome and the prevalence in Iceland we collected samples and information from all living individuals with a clinical diagnosis of Marfan syndrome in Iceland (n = 35) and performed whole-genome sequencing of those who did not have a confirmed genetic diagnosis. Moreover, to assess a potential underdiagnosis of Marfan syndrome in Iceland we attempted a genotype-based approach for identifying individuals with Marfan syndrome. We interrogated deCODE genetics’ database of 35,712 whole-genome sequenced individuals to search for rare sequence variants in FBN1. Overall, we identified 15 pathogenic or likely pathogenic variants in FBN1 in 41 living individuals, only 22 of whom were previously diagnosed with Marfan syndrome. The most common of these variants, NM_000138.4:c.8038C > T (p.Arg2680Cys), is present in a multi-generational pedigree, and was found to stem from a single forefather born around 1840. The p.Arg2680Cys associates with a form of Marfan syndrome that seems to have an enrichment of abdominal aortic aneurysm, suggesting that this may be a particularly common feature of p.Arg2680Cys-associated Marfan syndrome. Based on these combined genetic and clinical data, we estimate a Marfan syndrome prevalence of at least 1/6,000 in Iceland, compared to 1/10,000 based on clinical diagnosis alone, which indicates underdiagnosis of this actionable genetic disorder.

Age-related hearing impairment (ARHI) is the most common sensory disorder in older adults. We conducted a genome-wide association meta-analysis of 121,934 ARHI cases and 591,699 controls from Iceland and the UK. We identified 21 novel sequence variants, of which 13 are rare, under either additive or recessive models. Of special interest are a missense variant in LOXHD1 (MAF = 1.96%) and a tandem duplication in FBF1 covering 4 exons (MAF = 0.22%) associating with ARHI (OR = 3.7 for homozygotes, P = 1.7 × 10−22 and OR = 4.2 for heterozygotes, P = 5.7 × 10−27, respectively). We constructed an ARHI genetic risk score (GRS) using common variants and showed that a common variant GRS can identify individuals at risk comparable to carriers of rare high penetrance variants. Furthermore, we found that ARHI and tinnitus share genetic causes. This study sheds a new light on the genetic architecture of ARHI, through several rare variants in both Mendelian deafness genes and genes not previously linked to hearing., Erna Ivarsdottir et al. report a genome-wide association meta-analysis for age-related hearing loss in the Icelandic and UK populations. They identify 21 novel variants, 13 of which are rare, and reveal a genetic correlation between age-related hearing loss and tinnitus.

Long-read sequencing (LRS) promises to improve the characterization of structural variants (SVs). We generated LRS data from 3,622 Icelanders and identified a median of 22,636 SVs per individual (a median of 13,353 insertions and 9,474 deletions). We discovered a set of 133,886 reliably genotyped SV alleles and imputed them into 166,281 individuals to explore their effects on diseases and other traits. We discovered an association of a rare deletion in PCSK9 with lower low-density lipoprotein (LDL) cholesterol levels, compared to the population average. We also discovered an association of a multiallelic SV in ACAN with height; we found 11 alleles that differed in the number of a 57-bp-motif repeat and observed a linear relationship between the number of repeats carried and height. These results show that SVs can be accurately characterized at the population scale using LRS data in a genome-wide non-targeted approach and demonstrate how SVs impact phenotypes.

The characteristic lobulated nuclear morphology of granulocytes is partially determined by composition of nuclear envelope proteins. Abnormal nuclear morphology is primarily observed as an increased number of hypolobulated immature neutrophils, called band cells, during infection or in rare envelopathies like Pelger-Huët anomaly. To search for sequence variants affecting nuclear morphology of granulocytes, we performed a genome-wide association study using band neutrophil fraction from 88,101 Icelanders. We describe 13 sequence variants affecting band neutrophil fraction at nine loci. Five of the variants are at the Lamin B receptor (LBR) locus, encoding an inner nuclear membrane protein. Mutations in LBR are linked to Pelger-Huët anomaly. In addition, we identify cosegregation of a rare stop-gain sequence variant in LBR and Pelger Huët anomaly in an Icelandic eight generation pedigree, initially reported in 1963. Two of the other loci include genes which, like LBR, play a role in the nuclear membrane function and integrity. These GWAS results highlight the role proteins of the inner nuclear membrane have as important for neutrophil nuclear morphology.

Despite the important role that monozygotic twins have played in genetics research, little is known about their genomic differences. Here we show that monozygotic twins differ on average by 5.2 early developmental mutations and that approximately 15% of monozygotic twins have a substantial number of these early developmental mutations specific to one of them. Using the parents and offspring of twins, we identified pre-twinning mutations. We observed instances where a twin was formed from a single cell lineage in the pre-twinning cell mass and instances where a twin was formed from several cell lineages. CpG>TpG mutations increased in frequency with embryonic development, coinciding with an increase in DNA methylation. Our results indicate that allocations of cells during development shapes genomic differences between monozygotic twins.

Predicting the pathogenicity of biallelic missense variants can be challenging. Here, we use a deficit of observed homozygous carriers of missense variants, versus an expected number in a set of 153,054 chip-genotyped Icelanders, to identify potentially pathogenic genotypes. We follow three missense variants with a complete deficit of homozygosity and find that their pathogenic effect in homozygous state ranges from severe childhood disease to early embryonic lethality. One of these variants is in CPSF3, a gene not previously linked to disease. From a set of clinically sequenced Icelanders, and by sequencing archival samples targeted through the Icelandic genealogy, we find four homozygous carriers. Additionally, we find two homozygous carriers of Mexican descent of another missense variant in CPSF3. All six homozygous carriers of missense variants in CPSF3 show severe intellectual disability, seizures, microcephaly, and abnormal muscle tone. Here, we show how the absence of certain homozygous genotypes from a large population set can elucidate causes of previously unexplained recessive diseases and early miscarriage.

The spread of SARS-CoV-2 is dependent on several factors, both biological and behavioral. The effectiveness of various non-pharmaceutical interventions can largely be attributed to changes in human behavior, but quantifying this effect remains challenging. Reconstructing the transmission tree of the third wave of SARS-CoV-2 infections in Iceland using contact tracing and viral sequence data from 2522 cases enables us to compare the infectiousness of distinct groups of persons directly. We find that people diagnosed outside of quarantine are 89% more infectious than those diagnosed while in quarantine, and infectiousness decreases as a function of the time spent in quarantine. Furthermore, we find that people of working age, 16-66 years old, are 47% more infectious than those outside that age range. Lastly, the transmission tree enables us to model the effect that given population prevalence of vaccination would have had on the third wave had they been administered before that time using several different strategies. We find that vaccinating in order of ascending age or uniformly at random would have prevented more infections per vaccination than vaccinating in order of descending age.

De novo mutations (DNMs) cause a large proportion of severe rare diseases of childhood. DNMs that occur early may result in mosaicism of both somatic and germ cells. Such early mutations can cause recurrence of disease. We scanned 1,007 sibling pairs from 251 families and identified 878 DNMs shared by siblings (ssDNMs) at 448 genomic sites. We estimated DNM recurrence probability based on parental mosaicism, sharing of DNMs among siblings, parent-of-origin, mutation type and genomic position. We detected 57.2% of ssDNMs in the parental blood. The recurrence probability of a DNM decreases by 2.27% per year for paternal DNMs and 1.78% per year for maternal DNMs. Maternal ssDNMs are more likely to be T>C mutations than paternal ssDNMs, and less likely to be C>T mutations. Depending on the properties of the DNM, the recurrence probability ranges from 0.011% to 28.5%. We have launched an online calculator to allow estimation of DNM recurrence probability for research purposes.

Publisher's version (útgefin grein) Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations., Mutations in genes encoding subunits of the phagocyte NADPH oxidase complex are recognized to cause chronic granulomatous disease (CGD), a severe primary immunodeficiency. Here we describe how deficiency of CYBC1, a previously uncharacterized protein in humans (C17orf62), leads to reduced expression of NADPH oxidase’s main subunit (gp91phox) and results in CGD. Analyzing two brothers diagnosed with CGD we identify a homozygous loss-of-function mutation, p.Tyr2Ter, in CYBC1. Imputation of p.Tyr2Ter into 155K chipgenotyped Icelanders reveals six additional homozygotes, all with signs of CGD, manifesting as colitis, rare infections, or a severely impaired PMA-induced neutrophil oxidative burst. Homozygosity for p.Tyr2Ter consequently associates with inflammatory bowel disease (IBD) in Iceland (P = 8.3 × 10−8; OR = 67.6), as well as reduced height (P = 3.3 × 10−4; −8.5 cm). Overall, we find that CYBC1 deficiency results in CGD characterized by colitis and a distinct profile of infections indicative of macrophage dysfunction., We wish to thank the family of the two probands, as well as all the other individuals who participated in the study and whose contribution made this work possible.

Publisher's version (útgefin grein), Hemoglobin is the essential oxygen-carrying molecule in humans and is regulated by cellular iron and oxygen sensing mechanisms. To search for novel variants associated with hemoglobin concentration, we performed genome-wide association studies of hemoglobin concentration using a combined set of 684,122 individuals from Iceland and the UK. Notably, we found seven novel variants, six rare coding and one common, at the ACO1 locus associating with either decreased or increased hemoglobin concentration. Of these variants, the missense Cys506Ser and the stop-gained Lys334Ter mutations are specific to eight and ten generation pedigrees, respectively, and have the two largest effects in the study (EffectCys506Ser = −1.61 SD, CI95 = [−1.98, −1.35]; EffectLys334Ter = 0.63 SD, CI95 = [0.36, 0.91]). We also find Cys506Ser to associate with increased risk of persistent anemia (OR = 17.1, P = 2 × 10−14). The strong bidirectional effects seen in this study implicate ACO1, a known iron sensing molecule, as a major homeostatic regulator of hemoglobin concentration., We thank the individuals who participated in this study and whose contributions made this work possible. We also thank our valued colleagues who contributed to the data collection and phenotypic characterization of clinical samples as well as to the genotyping and analysis of the whole-genome association data. This research has been conducted using the UK Biobank Resource under application number 24711. F.W.A. is supported by UCL Hospitals NIHR Biomedical Research Centre.

Long-read sequencing (LRS) promises to improve characterization of structural variants (SVs), a major source of genetic diversity. We generated LRS data on 3,622 Icelanders using Oxford Nanopore Technologies, and identified a median of 22,636 SVs per individual (a median of 13,353 insertions and 9,474 deletions), spanning a median of 10 Mb per haploid genome. We discovered a set of 133,886 reliably genotyped SV alleles and imputed them into 166,281 individuals to explore their effects on diseases and other traits. We discovered an association with a rare (AF = 0.037%) deletion of the first exon ofPCSK9. Carriers of this deletion have 0.93 mmol/L (1.31 SD) lower LDL cholesterol levels than the population average (p-value = 7.0·10−20). We also discovered an association with a multi-allelic SV inside a large repeat region, contained within single long reads, in an exon ofACAN. Within this repeat region we found 11 alleles that differ in the number of a 57 bp-motif repeat, and observed a linear relationship (0.016 SD per motif inserted, p = 6.2·10−18) between the number of repeats carried and height. These results show that SVs can be accurately characterized at population scale using long read sequence data in a genome-wide non-targeted approach and demonstrate how SVs impact phenotypes.

Long-read sequencing (LRS) promises to improve the characterization of structural variants (SVs). We generated LRS data from 3,622 Icelanders and identified a median of 22,636 SVs per individual (a median of 13,353 insertions and 9,474 deletions). We discovered a set of 133,886 reliably genotyped SV alleles and imputed them into 166,281 individuals to explore their effects on diseases and other traits. We discovered an association of a rare deletion in PCSK9 with lower low-density lipoprotein (LDL) cholesterol levels, compared to the population average. We also discovered an association of a multiallelic SV in ACAN with height; we found 11 alleles that differed in the number of a 57-bp-motif repeat and observed a linear relationship between the number of repeats carried and height. These results show that SVs can be accurately characterized at the population scale using LRS data in a genome-wide non-targeted approach and demonstrate how SVs impact phenotypes.

Despite the important role that monozygotic twins have played in genetics research, little is known about their genomic differences. Here we show that monozygotic twins differ on average by 5.2 early developmental mutations and that approximately 15% of monozygotic twins have a substantial number of these early developmental mutations specific to one of them. Using the parents and offspring of twins, we identified pre-twinning mutations. We observed instances where a twin was formed from a single cell lineage in the pre-twinning cell mass and instances where a twin was formed from several cell lineages. CpGTpG mutations increased in frequency with embryonic development, coinciding with an increase in DNA methylation. Our results indicate that allocations of cells during development shapes genomic differences between monozygotic twins.

A fundamental requirement for genetic studies is an accurate determination of sequence variation. While human genome sequence diversity is increasingly well characterized, there is a need for efficient ways to use this knowledge in sequence analysis. Here we present Graphtyper, a publicly available novel algorithm and software for discovering and genotyping sequence variants. Graphtyper realigns short-read sequence data to a pangenome, a variation-aware graph structure that encodes sequence variation within a population by representing possible haplotypes as graph paths. Our results show that Graphtyper is fast, highly scalable, and provides sensitive and accurate genotype calls. Graphtyper genotyped 89.4 million sequence variants in the whole genomes of 28,075 Icelanders using less than 100,000 CPU days, including detailed genotyping of six human leukocyte antigen (HLA) genes. We show that Graphtyper is a valuable tool in characterizing sequence variation in both small and population-scale sequencing studies.

The characterization of mutational processes that generate sequence diversity in the human genome is of paramount importance both to medical genetics and to evolutionary studies. To understand how the age and sex of transmitting parents affect de novo mutations, here we sequence 1,548 Icelanders, their parents, and, for a subset of 225, at least one child, to 35× genome-wide coverage. We find 108,778 de novo mutations, both single nucleotide polymorphisms and indels, and determine the parent of origin of 42,961. The number of de novo mutations from mothers increases by 0.37 per year of age (95% CI 0.32-0.43), a quarter of the 1.51 per year from fathers (95% CI 1.45-1.57). The number of clustered mutations increases faster with the mother's age than with the father's, and the genomic span of maternal de novo mutation clusters is greater than that of paternal ones. The types of de novo mutation from mothers change substantially with age, with a 0.26% (95% CI 0.19-0.33%) decrease in cytosine-phosphate-guanine to thymine-phosphate-guanine (CpG>TpG) de novo mutations and a 0.33% (95% CI 0.28-0.38%) increase in C>G de novo mutations per year, respectively. Remarkably, these age-related changes are not distributed uniformly across the genome. A striking example is a 20 megabase region on chromosome 8p, with a maternal C>G mutation rate that is up to 50-fold greater than the rest of the genome. The age-related accumulation of maternal non-crossover gene conversions also mostly occurs within these regions. Increased sequence diversity and linkage disequilibrium of C>G variants within regions affected by excess maternal mutations indicate that the underlying mutational process has persisted in humans for thousands of years. Moreover, the regional excess of C>G variation in humans is largely shared by chimpanzees, less by gorillas, and is almost absent from orangutans. This demonstrates that sequence diversity in humans results from evolving interactions between age, sex, mutation type, and genomic location.

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.190 + 1G > C, minor allele frequency = 0.56%). 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 (P = 2.1 × 10-54), higher levels of non-high density lipoprotein cholesterol (β = 0.26 mmol/l, P = 2.6 × 10-9) and greater risk of coronary artery disease (odds ratio = 1.30, 95% confidence interval: 1.10-1.54, P = 0.0024). 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.

Publisher's version (útgefin grein), Background: Lipoprotein(a) [Lp(a)] is a causal risk factor for cardiovascular diseases that has no established therapy. The attribute of Lp(a) that affects cardiovascular risk is not established. Low levels of Lp(a) have been associated with type 2 diabetes (T2D). Objectives: This study investigated whether cardiovascular risk is conferred by Lp(a) molar concentration or apolipoprotein(a) [apo(a)] size, and whether the relationship between Lp(a) and T2D risk is causal. Methods: This was a case-control study of 143,087 Icelanders with genetic information, including 17,715 with coronary artery disease (CAD) and 8,734 with T2D. This study used measured and genetically imputed Lp(a) molar concentration, kringle IV type 2 (KIV-2) repeats (which determine apo(a) size), and a splice variant in LPA associated with small apo(a) but low Lp(a) molar concentration to disentangle the relationship between Lp(a) and cardiovascular risk. Loss-of-function homozygotes and other subjects genetically predicted to have low Lp(a) levels were evaluated to assess the relationship between Lp(a) and T2D. Results: Lp(a) molar concentration was associated dose-dependently with CAD risk, peripheral artery disease, aortic valve stenosis, heart failure, and lifespan. Lp(a) molar concentration fully explained the Lp(a) association with CAD, and there was no residual association with apo(a) size. Homozygous carriers of loss-of-function mutations had little or no Lp(a) and increased the risk of T2D. Conclusions: Molar concentration is the attribute of Lp(a) that affects risk of cardiovascular diseases. Low Lp(a) concentration (bottom 10%) increases T2D risk. Pharmacologic reduction of Lp(a) concentration in the 20% of individuals with the greatest concentration down to the population median is predicted to decrease CAD risk without increasing T2D risk.

Dilated cardiomyopathy (DCM) is an important cause of heart failure. Variants in50 genes have been reported to cause DCM, but causative variants have been found in less than half of familial cases. Variants causing DCM in Iceland have not been reported before.We performed a genome-wide association study on DCM based on whole genome sequencing. We tested the association of 32.5 million sequence variants in 424 cases and 337 689 population controls in Iceland.We identified 2 DCM variants in established cardiomyopathy genes, a missense variant p.Phe145Leu in NKX2-5 carried by 1 in 7100 Icelanders ( P=7.0×10Two rare variants in NKX2-5 and FLNC, carried by 1 in 2400 Icelanders, cause familial DCM in Iceland. These genes have recently been associated with DCM. Given the serious consequences of these variants, we suggest screening for them in individuals with DCM and their family members, with subsequent monitoring of carriers, offering early intervention.

Meiotic recombination involves a combination of gene conversion and crossover events that, along with mutations, produce germline genetic diversity. Here we report the discovery of 3,176 SNP and 61 indel gene conversions. Our estimate of the non-crossover (NCO) gene conversion rate (G) is 7.0 for SNPs and 5.8 for indels per megabase per generation, and the GC bias is 67.6%. For indels, we demonstrate a 65.6% preference for the shorter allele. NCO gene conversions from mothers are longer than those from fathers, and G is 2.17 times greater in mothers. Notably, G increases with the age of mothers, but not the age of fathers. A disproportionate number of NCO gene conversions in older mothers occur outside double-strand break (DSB) regions and in regions with relatively low GC content. This points to age-related changes in the mechanisms of meiotic gene conversion in oocytes.

Meiotic recombination involves a combination of gene conversion and crossover events that along with mutations produce germline genetic diversity. Here, we report the discovery of 3,176 SNP and 61 indel gene conversions. Our estimate of the non-crossover (NCO) gene conversion rate (G) is 7.0 for SNPs and 5.8 for indels per Mb per generation, and the GC bias is 67.6%. For indels we demonstrate a 65.6% preference for the shorter allele. NCO gene conversions from mothers are longer than those from fathers and G is 2.17 times greater in mothers. Notably, G increases with the age of mothers, but not fathers. A disproportionate number of NCO gene conversions in older mothers occur outside double strand break (DSB) regions and in regions with relatively low GC content. This points to age-related changes in the mechanisms of meiotic gene conversions in oocytes.

Publisher's version (útgefin grein). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations., Discovery of coding variants in genes that confer risk of neurodevelopmental disorders is an important step towards understanding the pathophysiology of these disorders. Wholegenome sequencing of 31,463 Icelanders uncovers a frameshift variant (E712KfsTer10) in microtubule-associated protein 1B (MAP1B) that associates with ID/low IQ in a large pedigree (genome-wide corrected P = 0.022). Additional stop-gain variants in MAP1B (E1032Ter and R1664Ter) validate the association with ID and IQ. Carriers have 24% less white matter (WM) volume (β = −2.1SD, P = 5.1 × 10−8), 47% less corpus callosum (CC) volume (β = −2.4SD, P = 5.5 × 10−10) and lower brain-wide fractional anisotropy (P = 6.7 × 10−4). In summary, we show that loss of MAP1B function affects general cognitive ability through a profound, brain-wide WM deficit with likely disordered or compromised axons., We are grateful to the participants and we thank the psychologists, nurses and staff, in particular Berglind Eiriksdottir, at the Research Recruitment Center and technicians and staff at Röntgen Domus. We also thank the staff at deCODE genetics core facilities and all our colleagues for their important contribution to this work. L.J. received support from the Swedish Society of Medicine, the Swedish Brain Foundation and Swedish Society for Medical Research. The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreements’ no. 115008 (NEWMEDS) and no. 115300 (EUAIMS) of which resources are composed of EFPIA in-kind contribution and financial contribution from the European Union’s Seventh Framework Programme (EU-FP7/2007-2013), EU-FP7 funded grant no. 602450 (IMAGEMEND) and EU funded FP7-People-2011-IAPP grant agreement no. 286213 (PsychDPC).

Searching for novel sequence variants associated with cholesterol levels is of particular interest due to the causative role of non-HDL cholesterol levels in cardiovascular disease. Through whole-genome sequencing of 15,220 Icelanders and imputation of the variants identified, we discovered a rare missense variant in NR1H4 (R436H) associating with lower levels of total cholesterol (effect = −0.47 standard deviations or −0.55 mmol L−1, p = 4.21 × 10−10, N = 150,211). Importantly, NR1H4 R436H also associates with lower levels of non-HDL cholesterol and, consistent with this, protects against coronary artery disease. NR1H4 encodes FXR that regulates bile acid homeostasis, however, we do not detect a significant association between R436H and biological markers of liver function. Transcriptional profiling of hepatocytes carrying R436H shows that it is not a loss-of-function variant. Rather, we observe changes in gene expression compatible with effects on lipids. These findings highlight the role of FXR in regulation of cholesterol levels in humans. Aimee Deaton et al. identify a rare missense variant in the bile acid receptor gene NR1H4, which is associated with lower levels of total cholesterol in the Icelandic population. Hepatocytes expressing the missense variant showed altered expression of a small number of genes, with enrichment in lipid-related pathways.

Searching for novel sequence variants associated with cholesterol levels is of particular interest due to the causative role of non-HDL cholesterol levels in cardiovascular disease. Through whole-genome sequencing of 15,220 Icelanders and imputation of the variants identified, we discovered a rare missense variant in

De novo mutations (DNMs) cause a large proportion of severe rare diseases of childhood. DNMs that occur early may result in mosaicism of both somatic and germ cells. Such early mutations can cause recurrence of disease. We scanned 1,007 sibling pairs from 251 families and identified 878 DNMs shared by siblings (ssDNMs) at 448 genomic sites. We estimated DNM recurrence probability based on parental mosaicism, sharing of DNMs among siblings, parent-of-origin, mutation type and genomic position. We detected 57.2% of ssDNMs in the parental blood. The recurrence probability of a DNM decreases by 2.27% per year for paternal DNMs and 1.78% per year for maternal DNMs. Maternal ssDNMs are more likely to be TC mutations than paternal ssDNMs, and less likely to be CT mutations. Depending on the properties of the DNM, the recurrence probability ranges from 0.011% to 28.5%. We have launched an online calculator to allow estimation of DNM recurrence probability for research purposes.

Background: Rare missense mutations in the gene encoding coatomer subunit alpha (COPA) have recently been shown to cause autoimmune interstitial lung, joint and kidney disease, also known as COPA syndrome, under a dominant mode of inheritance. Case presentation: Here we describe an Icelandic family with three affected individuals over two generations with a rare clinical presentation of lung and joint disease and a histological diagnosis of follicular bronchiolitis. We performed whole-genome sequencing (WGS) of the three affected as well as three unaffected members of the family, and searched for rare genotypes associated with disease using 30,067 sequenced Icelanders as a reference population. We assessed all coding and splicing variants, prioritizing variants in genes known to cause interstitial lung disease. We detected a heterozygous missense mutation, p.Glu241Lys, in the COPA gene, private to the affected family members. The mutation occurred de novo in the paternal germline of the index case and was absent from 30,067 Icelandic genomes and 141,353 individuals from the genome Aggregation Database (gnomAD). The mutation occurs within the conserved and functionally important WD40 domain of the COPA protein. Conclusions: This is the second report of the p.Glu241Lys mutation in COPA, indicating the recurrent nature of the mutation. The mutation was reported to co-segregate with COPA syndrome in a large family from the USA with five affected members, and classified as pathogenic. The two separate occurrences of the p.Glu241Lys mutation in cases and its absence from a large number of sequenced genomes confirms its role in the pathogenesis of the COPA syndrome. Keywords: COPA syndrome, Lung disease, Arthritis, Immune dysregulation, Case report

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. Electronic supplementary material The online version of this article (10.1186/s12881-017-0466-8) contains supplementary material, which is available to authorized users.

Gastric cancer is a serious health problem worldwide, with particularly high prevalence in eastern Asia. Genome-wide association studies (GWAS) in Asian populations have identified several loci that associate with gastric cancer risk. Here we report a GWAS of gastric cancer in a European population, using information on 2,500 population-based gastric cancer cases and 205,652 controls. We found a new gastric cancer association with loss-of-function mutations in ATM (gene test, P = 8.0 × 10-12; odds ratio (OR) = 4.74). The combination of the loss-of-function variants p.Gln852∗, p.Ser644∗ and p.Tyr103∗ (combined minor allele frequency (MAF) = 0.3%) also associates with pancreatic and prostate cancers (OR = 3.81 and 2.18, respectively) and gives an indication of risk of breast and colorectal cancers (OR = 1.82 and 1.97, respectively). Cancers in those carrying loss-of-function ATM mutations are diagnosed at a significantly earlier age than in non-carriers. Our results confirm an association between gastric cancer in Europeans and three loci previously reported in Asians, MUC1, PRKAA1 and PSCA, refine the association signal at PRKAA1 and support a pathogenic role for the tandem repeat identified in MUC1.

Understanding of sequence diversity is the cornerstone of analysis of genetic disorders, population genetics, and evolutionary biology. Here, we present an update of our sequencing set to 15,220 Icelanders who we sequenced to an average genome-wide coverage of 34X. We identified 39,020,168 autosomal variants passing GATK filters: 31,079,378 SNPs and 7,940,790 indels. Calling de novo mutations (DNMs) is a formidable challenge given the high false positive rate in sequencing datasets relative to the mutation rate. Here we addressed this issue by using segregation of alleles in three-generation families. Using this transmission assay, we controlled the false positive rate and identified 108,778 high quality DNMs. Furthermore, we used our extended family structure and read pair tracing of DNMs to a panel of phased SNPs, to determine the parent of origin of 42,961 DNMs.

A fundamental requisite for genetic studies is an accurate determination of sequence variation. While human genome sequence diversity is increasingly well characterized, there is a need for efficient ways to utilize this knowledge in sequence analysis. Here we present Graphtyper, a publicly available novel algorithm and software for discovering and genotyping sequence variants. Graphtyper realigns short-read sequence data to a pangenome, a variation-aware graph structure that encodes sequence variation within a population by representing possible haplotypes as graph paths. Our results show that Graphtyper is fast, highly scalable, and provides sensitive and accurate genotype calls. Graphtyper genotyped 89.4 million sequence variants in whole-genomes of 28,075 Icelanders using less than 100,000 CPU days, including detailed genotyping of six human leukocyte antigen (HLA) genes. We show that Graphtyper is a valuable tool in characterizing sequence variation in population-scale sequencing studies.

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., The Dutch asthma study was supported by the Netherlands Asthma Foundation grant AF (AF 95.09, AF 98.48, AF 3.2.02.51 and AF 3.2.07.015) and a grant from the University Medical Center Groningen. The Vlagtwedde-Vlaardingen cohort study was supported by the Ministry of Health and Environmental Hygiene of the Netherlands and the Netherlands Asthma Fund (grant 187) and the Netherlands Asthma Fund grant no. 3.2.02.51, the Stichting Astma Bestrijding, BBMRI-NL (Complementiation project), and the European Respiratory Society COPD research award 2011 to H.M. Boezen. COPSAC is funded by private and public research funds all listed on www.copsac.com. The Lundbeck Foundation; Danish State Budget; Danish Council for Strategic Research; Danish Council for Independent Research and The Capital Region Research Foundation have provided core support for COPSAC. The Denmark-1 study has been supported with an unrestricted grant from AstraZeneca and ALK-Abello, Denmark. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Pulmonary function test results from the three affected Icelanders with several years passing between the two tests. (DOCX 13Â kb)

Additional clinical features. Autoimmune and rheumatological features as well as antibody titers for the three patients. The medications and general measures taken to treat the patients are listed. (DOCX 13Â kb)

COPA mutation. Summary of the causative mutation causing the lung disease in the Icelandic family. (DOCX 19Â kb)

Coding variants with MAFÂ

Lung biopsy section from the daughter of the index case (III-2) stained with hematoxylin and eosin. Multiple lymphoid follicles in the interstitium and adjacent to airways (arrow). (DOCX 856Â kb)

Pulmonary function test results from the three affected Icelanders with several years passing between the two tests. (DOCX 13Â kb)

Additional clinical features. Autoimmune and rheumatological features as well as antibody titers for the three patients. The medications and general measures taken to treat the patients are listed. (DOCX 13Â kb)

Detailed description of methods (sample preparation, whole-genome sequencing, alignment, variant calling and annotation) and the genetic analysis performed in this study (Additional file 10: Table S5). (DOCX 23 kb)

COPA mutation. Summary of the causative mutation causing the lung disease in the Icelandic family. (DOCX 19Â kb)

Lung biopsy section from the son of the index case (III-1) stained with hematoxylin and eosin. Multiple lymphoid follicles in the pulmonary interstitium and adjacent to a terminal bronchiole (arrow). (DOCX 906Â kb)

Variant filtering flowchart. We detected an average of 4,942,809 coding and non-coding variants per affected member of the family. Sixteen variants with MAF

Variant filtering flowchart. We detected an average of 4,942,809 coding and non-coding variants per affected member of the family. Sixteen variants with MAF

Sequencing data metrics for the COPA mutation. The call ratios of the COPA mutation for the index case, affected son and daughter, and unaffected husband, mother, and father from the WGS data. Additionally, the Sanger sequencing genotypes for the three siblings of the index case are listed. (DOCX 13Â kb)

Lung biopsy section from the daughter of the index case (III-2) stained with hematoxylin and eosin. Multiple lymphoid follicles in the interstitium and adjacent to airways (arrow). (DOCX 856Â kb)

Lung biopsy section from the son of the index case (III-1) stained with hematoxylin and eosin. Multiple lymphoid follicles in the pulmonary interstitium and adjacent to a terminal bronchiole (arrow). (DOCX 906Â kb)

Sequencing data metrics for the COPA mutation. The call ratios of the COPA mutation for the index case, affected son and daughter, and unaffected husband, mother, and father from the WGS data. Additionally, the Sanger sequencing genotypes for the three siblings of the index case are listed. (DOCX 13Â kb)

Chronic kidney disease (CKD) is a complex disorder with a strong genetic component. A number of common sequence variants have been found to associate with serum creatinine (SCr), estimated glomerular filtration rate (eGFR) and/or CKD. We imputed 24 million single-nucleotide polymorphisms and insertions/deletions identified by whole-genome sequencing of 2230 Icelanders into 81 656 chip-typed individuals and 112 630 relatives of genotyped individuals over the age of 18 with SCr measurements. The large set of sequenced individuals allowed accurate imputation of variants to a minor allele frequency (MAF) of 0.1%. We tested the imputed variants for association with SCr. In addition to replicating established loci, we discovered missense and loss-of-function variants associating with SCr in three solute carriers (SLC6A19, SLC25A45 and SLC47A1) and two E3 ubiquitin ligases (RNF186 and RNF128). All the variants are within coding sequences and all but one are rare (MAF

Mutation of the DNA molecule is one of the most fundamental processes in biology. In this study, we use 283 parent-offspring trios to estimate the rate of mutation for both single nucleotide variants (SNVs) and short length variants (indels) in humans and examine the mutation process. We found 17812 SNVs, corresponding to a mutation rate of 1.29 × 10−8 per position per generation (PPPG) and 1282 indels corresponding to a rate of 9.29 × 10−10 PPPG. We estimate that around 3% of human de novo SNVs are part of a multi-nucleotide mutation (MNM), with 558 (3.1%) of mutations positioned less than 20kb from another mutation in the same individual (median distance of 525bp). The rate of de novo mutations is greater in late replicating regions (p = 8.29 × 10−19) and nearer recombination events (p = 0.0038) than elsewhere in the genome., Author Summary In each generation new genetic variants are introduced by mutations. In this study we use whole genome sequence data from Icelandic families to directly observe such new mutations. Our estimate of the mutation rate implies that a newborn with 30-year-old parents will on average carry 75 new SNV mutations and 6 new short indel mutations. We observe that some of the found mutations occur much closer together than would be expected by chance. Our analysis shows that mutational hotspots cannot explain this clustering, instead the clustering mutations are likely created by a single mutational event. We observe a different composition for mutations that cluster very close together compared to more distant clustering mutations. This suggests that there is likely more than one type of underlying mutational mechanism creating the multi nucleotide mutation events. We furthermore observe a higher mutation rate near recombination events but find that this effect cannot explain the large number of clustering mutations that we observe.

Genomes usually contain some non-repetitive sequences that are missing from the reference genome and occur only in a population subset. Such non-repetitive, non-reference (NRNR) sequences have remained largely unexplored in terms of their characterization and downstream analyses. Here we describe 3,791 breakpoint-resolved NRNR sequence variants called using PopIns from whole-genome sequence data of 15,219 Icelanders. We found that over 95% of the 244 NRNR sequences that are 200 bp or longer are present in chimpanzees, indicating that they are ancestral. Furthermore, 149 variant loci are in linkage disequilibrium (r2 > 0.8) with a genome-wide association study (GWAS) catalog marker, suggesting disease relevance. Additionally, we report an association (P = 3.8 × 10-8, odds ratio (OR) = 0.92) with myocardial infarction (23,360 cases, 300,771 controls) for a 766-bp NRNR sequence variant. Our results underline the importance of including variation of all complexity levels when searching for variants that associate with disease.

Analysis of whole-genome sequence data of Icelandic individuals has revealed a rare nonsense mutation within the LGR4 gene that is strongly associated with, among other things, low bone mineral density, late onset of menarche, and increased risk of biliary tract cancer. A search through whole-genome sequence data from thousands of Icelandic individuals for variants that may have a direct effect on the risk of pathologically low bone mineral density has identified a rare nonsense mutation that associates with a wide range of phenotypes in addition to those related to bone physiology. The mutation, found within the leucine-rich-repeat-containing G-protein-coupled receptor 4 (LGR4) gene, is strongly associated with osteoporotic fractures, and also with electrolyte imbalance, hormonal imbalance and an increased risk of squamous cell carcinoma of the skin and biliary tract cancer. The phenotype of carriers of this mutation overlaps with that of Lgr4 knockout mice. 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 BMD1,2,3,4,5,6,7. 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.