Evaluating the clinical validity of genes related to hemostasis and thrombosis using the Clinical Genome Resource gene curation framework.

BACKGROUND: Inherited bleeding, thrombotic, and platelet disorders (BTPDs) are a heterogeneous set of diseases, many of which are very rare globally. Over the past 5 decades, the genetic basis of some of these disorders has been identified, and recently, high-throughput sequencing has become the primary means of identifying disease-causing genetic variants. OBJECTIVES: Knowledge of the clinical validity of a gene-disease relationship is essential to provide an accurate diagnosis based on results of diagnostic gene panel tests and inform the construction of such panels. The Scientific and Standardization Committee for Genetics in Thrombosis and Hemostasis undertook a curation process for selecting 96 TIER1 genes for BTPDs. The purpose of the process was to evaluate the evidence supporting each gene-disease relationship and provide an expert-reviewed classification for the clinical validity of genes associated with BTPDs. METHODS: The Clinical Genome Resource (ClinGen) Hemostasis/Thrombosis Gene Curation Expert Panel assessed the strength of evidence for TIER1 genes using the semiquantitative ClinGen gene-disease clinical validity framework. ClinGen Lumping and Splitting guidelines were used to determine the appropriate disease entity or entities for each gene, and 101 gene-disease relationships were identified for curation. RESULTS: The final outcome included 68 Definitive (67%), 26 Moderate (26%), and 7 Limited (7%) classifications. The summary of each curation is available on the ClinGen website. CONCLUSION: Expert-reviewed assignment of gene-disease relationships by the ClinGen Hemostasis/Thrombosis Gene Curation Expert Panel facilitates accurate molecular diagnoses of BTPDs by clinicians and diagnostic laboratories. These curation efforts can allow genetic testing to focus on genes with a validated role in disease.

Genome sequencing and comprehensive rare-variant analysis of 465 families with neurodevelopmental disorders.

Despite significant progress in unraveling the genetic causes of neurodevelopmental disorders (NDDs), a substantial proportion of individuals with NDDs remain without a genetic diagnosis after microarray and/or exome sequencing. Here, we aimed to assess the power of short-read genome sequencing (GS), complemented with long-read GS, to identify causal variants in participants with NDD from the National Institute for Health and Care Research (NIHR) BioResource project. Short-read GS was conducted on 692 individuals (489 affected and 203 unaffected relatives) from 465 families. Additionally, long-read GS was performed on five affected individuals who had structural variants (SVs) in technically challenging regions, had complex SVs, or required distal variant phasing. Causal variants were identified in 36% of affected individuals (177/489), and a further 23% (112/489) had a variant of uncertain significance after multiple rounds of re-analysis. Among all reported variants, 88% (333/380) were coding nuclear SNVs or insertions and deletions (indels), and the remainder were SVs, non-coding variants, and mitochondrial variants. Furthermore, long-read GS facilitated the resolution of challenging SVs and invalidated variants of difficult interpretation from short-read GS. This study demonstrates the value of short-read GS, complemented with long-read GS, in investigating the genetic causes of NDDs. GS provides a comprehensive and unbiased method of identifying all types of variants throughout the nuclear and mitochondrial genomes in individuals with NDD.

The effects of pathogenic and likely pathogenic variants for inherited hemostasis disorders in 140 214 UK Biobank participants.

Rare genetic diseases affect millions, and identifying causal DNA variants is essential for patient care. Therefore, it is imperative to estimate the effect of each independent variant and improve their pathogenicity classification. Our study of 140 214 unrelated UK Biobank (UKB) participants found that each of them carries a median of 7 variants previously reported as pathogenic or likely pathogenic. We focused on 967 diagnostic-grade gene (DGG) variants for rare bleeding, thrombotic, and platelet disorders (BTPDs) observed in 12 367 UKB participants. By association analysis, for a subset of these variants, we estimated effect sizes for platelet count and volume, and odds ratios for bleeding and thrombosis. Variants causal of some autosomal recessive platelet disorders revealed phenotypic consequences in carriers. Loss-of-function variants in MPL, which cause chronic amegakaryocytic thrombocytopenia if biallelic, were unexpectedly associated with increased platelet counts in carriers. We also demonstrated that common variants identified by genome-wide association studies (GWAS) for platelet count or thrombosis risk may influence the penetrance of rare variants in BTPD DGGs on their associated hemostasis disorders. Network-propagation analysis applied to an interactome of 18 410 nodes and 571 917 edges showed that GWAS variants with large effect sizes are enriched in DGGs and their first-order interactors. Finally, we illustrate the modifying effect of polygenic scores for platelet count and thrombosis risk on disease severity in participants carrying rare variants in TUBB1 or PROC and PROS1, respectively. Our findings demonstrate the power of association analyses using large population datasets in improving pathogenicity classifications of rare variants.

Investigating genotype-phenotype relationship of extreme neuropathic pain disorders in a UK national cohort.

The aims of our study were to use whole genome sequencing in a cross-sectional cohort of patients to identify new variants in genes implicated in neuropathic pain, to determine the prevalence of known pathogenic variants and to understand the relationship between pathogenic variants and clinical presentation. Patients with extreme neuropathic pain phenotypes (both sensory loss and gain) were recruited from secondary care clinics in the UK and underwent whole genome sequencing as part of the National Institute for Health and Care Research Bioresource Rare Diseases project. A multidisciplinary team assessed the pathogenicity of rare variants in genes previously known to cause neuropathic pain disorders and exploratory analysis of research candidate genes was completed. Association testing for genes carrying rare variants was completed using the gene-wise approach of the combined burden and variance-component test SKAT-O. Patch clamp analysis was performed on transfected HEK293T cells for research candidate variants of genes encoding ion channels. The results include the following: (i) Medically actionable variants were found in 12% of study participants (205 recruited), including known pathogenic variants: SCN9A(ENST00000409672.1): c.2544T>C, p.Ile848Thr that causes inherited erythromelalgia, and SPTLC1(ENST00000262554.2):c.340T>G, p.Cys133Tr variant that causes hereditary sensory neuropathy type-1. (ii) Clinically relevant variants were most common in voltage-gated sodium channels (Nav). (iii) SCN9A(ENST00000409672.1):c.554G>A, pArg185His variant was more common in non-freezing cold injury participants than controls and causes a gain of function of NaV1.7 after cooling (the environmental trigger for non-freezing cold injury). (iv) Rare variant association testing showed a significant difference in distribution for genes NGF, KIF1A, SCN8A, TRPM8, KIF1A, TRPA1 and the regulatory regions of genes SCN11A, FLVCR1, KIF1A and SCN9A between European participants with neuropathic pain and controls. (v) The TRPA1(ENST00000262209.4):c.515C>T, p.Ala172Val variant identified in participants with episodic somatic pain disorder demonstrated gain-of-channel function to agonist stimulation. Whole genome sequencing identified clinically relevant variants in over 10% of participants with extreme neuropathic pain phenotypes. The majority of these variants were found in ion channels. Combining genetic analysis with functional validation can lead to a better understanding as to how rare variants in ion channels lead to sensory neuron hyper-excitability, and how cold, as an environmental trigger, interacts with the gain-of-function NaV1.7 p.Arg185His variant. Our findings highlight the role of ion channel variants in the pathogenesis of extreme neuropathic pain disorders, likely mediated through changes in sensory neuron excitability and interaction with environmental triggers.

Effectiveness and costs of a stepwise versus an all-in-one approach to diagnose mild bleeding disorders.

The diagnostic work-up of patients referred to the haematologist for bleeding evaluation is performed in a stepwise way: bleeding history and results of screening laboratory tests guide further diagnostic evaluation. This can be ineffective, time-consuming and burdensome for patients. To improve this strategy, the initial laboratory investigation can be extended. In a model-based approach, effectiveness and costs of a conventional stepwise versus a newly proposed all-in-one diagnostic approach for bleeding evaluation were evaluated and compared, using data from an observational patient cohort study, including adult patients referred for bleeding evaluation. In the all-in-one approach, specialized platelet function tests, coagulation factors, and fibrinolysis tests were included in the initial investigation. Final diagnosis, hospital resource use and costs and patient burden were compared. A total of 150 patients were included. Compared to the stepwise approach, in the all-in-one approach, 19 additional patients reached a diagnosis and patient burden was lower, but total costs per patient were higher [€359, 95% bootstrapped confidence interval (BCI) 283-518, p = 0.001]. For bleeding evaluation of patients referred to the haematologist, an all-in-one diagnostic approach has a higher diagnostic yield and reduces patient burden, at a higher cost. This raises the question what costs justify the diagnosis of a bleeding disorder and a less burdensome diagnostic strategy.

Implementation of individualised polygenic risk score analysis: a test case of a family of four.

BACKGROUND: Polygenic risk scores (PRS) have been widely applied in research studies, showing how population groups can be stratified into risk categories for many common conditions. As healthcare systems consider applying PRS to keep their populations healthy, little work has been carried out demonstrating their implementation at an individual level. CASE PRESENTATION: We performed a systematic curation of PRS sources from established data repositories, selecting 15 phenotypes, comprising an excess of 37 million SNPs related to cancer, cardiovascular, metabolic and autoimmune diseases. We tested selected phenotypes using whole genome sequencing data for a family of four related individuals. Individual risk scores were given percentile values based upon reference distributions among 1000 Genomes Iberians, Europeans, or all samples. Over 96 billion allele effects were calculated in order to obtain the PRS for each of the individuals analysed here. CONCLUSIONS: Our results highlight the need for further standardisation in the way PRS are developed and shared, the importance of individual risk assessment rather than the assumption of inherited averages, and the challenges currently posed when translating PRS into risk metrics.

First Genotype-Phenotype Study in TBX4 Syndrome: Gain-of-Function Mutations Causative for Lung Disease.

Rationale: Despite the increased recognition of TBX4 (T-BOX transcription factor 4)-associated pulmonary arterial hypertension (PAH), genotype-phenotype associations are lacking and may provide important insights. Objectives: To compile and functionally characterize all TBX4 variants reported to date and undertake a comprehensive genotype-phenotype analysis. Methods: We assembled a multicenter cohort of 137 patients harboring monoallelic TBX4 variants and assessed the pathogenicity of missense variation (n = 42) using a novel luciferase reporter assay containing T-BOX binding motifs. We sought genotype-phenotype correlations and undertook a comparative analysis with patients with PAH with BMPR2 (Bone Morphogenetic Protein Receptor type 2) causal variants (n = 162) or no identified variants in PAH-associated genes (n = 741) genotyped via the National Institute for Health Research BioResource-Rare Diseases. Measurements and Main Results: Functional assessment of TBX4 missense variants led to the novel finding of gain-of-function effects associated with older age at diagnosis of lung disease compared with loss-of-function effects (P = 0.038). Variants located in the T-BOX and nuclear localization domains were associated with earlier presentation (P = 0.005) and increased incidence of interstitial lung disease (P = 0.003). Event-free survival (death or transplantation) was shorter in the T-BOX group (P = 0.022), although age had a significant effect in the hazard model (P = 0.0461). Carriers of TBX4 variants were diagnosed at a younger age (P < 0.001) and had worse baseline lung function (FEV1, FVC) (P = 0.009) than the BMPR2 and no identified causal variant groups. Conclusions: We demonstrated that TBX4 syndrome is not strictly the result of haploinsufficiency but can also be caused by gain of function. The pleiotropic effects of TBX4 in lung disease may be in part explained by the differential effect of pathogenic mutations located in critical protein domains.

Refinements and considerations for trio whole-genome sequence analysis when investigating Mendelian diseases presenting in early childhood.

To facilitate early deployment of whole-genome sequencing (WGS) for severely ill children, a standardized pipeline for WGS analysis with timely turnaround and primary care pediatric uptake is needed. We developed a bioinformatics pipeline for comprehensive gene-agnostic trio WGS analysis of children suspected of having an undiagnosed monogenic disease that included detection and interpretation of primary genetic mechanisms of disease, including SNVs/indels, CNVs/SVs, uniparental disomy (UPD), imprinted genes, short tandem repeat expansions, mobile element insertions, SMN1/2 copy number calling, and mitochondrial genome variants. We assessed primary care practitioner experience and competence in a large cohort of 521 families (comprising 90% WGS trios). Children were identified by primary practitioners for recruitment, and we used the UK index of multiple deprivation to confirm lack of patient socio-economic status ascertainment bias. Of the 521 children sequenced, 176 (34%) received molecular diagnoses, with rates as high as 45% for neurology clinics. Twenty-three of the diagnosed cases (13%) required bespoke methods beyond routine SNV/CNV analysis. In our multidisciplinary clinician user experience assessment, both pediatricians and clinical geneticists expressed strong support for rapid WGS early in the care pathway, but requested further training in determining patient selection, consenting, and variant interpretation. Rapid trio WGS provides an efficacious single-pass screening test for children when deployed by primary practitioners in clinical settings that carry high a priori risk for rare pediatric disease presentations.

Exploring the relevance of NUP93 variants in steroid-resistant nephrotic syndrome using next generation sequencing and a fly kidney model.

BACKGROUND: Variants in genes encoding nuclear pore complex (NPC) proteins are a newly identified cause of paediatric steroid-resistant nephrotic syndrome (SRNS). Recent reports describing NUP93 variants suggest these could be a significant cause of paediatric onset SRNS. We report NUP93 cases in the UK and demonstrate in vivo functional effects of Nup93 depletion in a fly (Drosophila melanogaster) nephrocyte model. METHODS: Three hundred thirty-seven paediatric SRNS patients from the National cohort of patients with Nephrotic Syndrome (NephroS) were whole exome and/or whole genome sequenced. Patients were screened for over 70 genes known to be associated with Nephrotic Syndrome (NS). D. melanogaster Nup93 knockdown was achieved by RNA interference using nephrocyte-restricted drivers. RESULTS: Six novel homozygous and compound heterozygous NUP93 variants were detected in 3 sporadic and 2 familial paediatric onset SRNS characterised histologically by focal segmental glomerulosclerosis (FSGS) and progressing to kidney failure by 12 months from clinical diagnosis. Silencing of the two orthologs of human NUP93 expressed in D. melanogaster, Nup93-1, and Nup93-2 resulted in significant signal reduction of up to 82% in adult pericardial nephrocytes with concomitant disruption of NPC protein expression. Additionally, nephrocyte morphology was highly abnormal in Nup93-1 and Nup93-2 silenced flies surviving to adulthood. CONCLUSION: We expand the spectrum of NUP93 variants detected in paediatric onset SRNS and demonstrate its incidence within a national cohort. Silencing of either D. melanogaster Nup93 ortholog caused a severe nephrocyte phenotype, signaling an important role for the nucleoporin complex in podocyte biology. A higher resolution version of the Graphical abstract is available as Supplementary information.

Multiparameter platelet function analysis of bleeding patients with a prolonged platelet function analyser closure time.

Patients referred for evaluation of bleeding symptoms occasionally have a prolonged platelet function analyser (PFA) closure time, without evidence for von Willebrand disease or impaired platelet aggregation. The aim of this study was to establish a shear-dependent platelet function defect in these patients. Patients were included based on high bleeding score and prior PFA prolongation. Common tests of von Willebrand factor (VWF) and platelet function and exome sequencing were performed. Microfluidic analysis of shear-dependent collagen-induced whole-blood thrombus formation was performed. In 14 PFA-only patients, compared to healthy volunteers, microfluidic tests showed significantly lower platelet adhesion and thrombus formation parameters. This was accompanied by lower integrin activation, phosphatidylserine exposure and P-selectin expression. Principal components analysis indicated VWF as primary explaining variable of PFA prolongation, whereas conventional platelet aggregation primarily explained the reduced thrombus parameters under shear. In five patients with severe microfluidic abnormalities, conventional platelet aggregation was in the lowest range of normal. No causal variants in Mendelian genes known to cause bleeding or platelet disorders were identified. Multiparameter assessment of whole-blood thrombus formation under shear indicates single or combined effects of low-normal VWF and low-normal platelet aggregation in these patients, suggesting a shear-dependent platelet function defect, not detected by static conventional haemostatic tests.

GoldVariants, a resource for sharing rare genetic variants detected in bleeding, thrombotic, and platelet disorders: Communication from the ISTH SSC Subcommittee on Genomics in Thrombosis and Hemostasis.

The implementation of high-throughput sequencing (HTS) technologies in research and diagnostic laboratories has linked many new genes to rare bleeding, thrombotic, and platelet disorders (BTPD), and revealed multiple genetic variants linked to those disorders, many of them being of uncertain pathogenicity when considering the accepted evidence (variant consequence, frequency in control datasets, number of reported patients, prediction models, and functional assays). The sequencing effort has also resulted in resources for gathering disease-causing variants associated with specific genes, but for BTPD, such well-curated databases exist only for a few genes. On the other hand, submissions by individuals or diagnostic laboratories to the variant database ClinVar are hampered by the lack of a submission process tailored to capture the specific features of hemostatic diseases. As we move toward the implementation of HTS in the diagnosis of BTPD, the Scientific and Standardization Committee for Genetics in Thrombosis and Haemostasis has developed and tested a REDCap-based interface, aimed at the community, to submit curated genetic variants for diagnostic-grade BTPD genes. Here, we describe the use of the interface and the initial submission of 821 variants from 30 different centers covering 14 countries. This open-access variant resource will be shared with the community to improve variant classification and regular bulk data transfer to ClinVar.

Advances in understanding the pathogenesis of hereditary macrothrombocytopenia.

Low platelet count, or thrombocytopenia, is a common haematological abnormality, with a wide differential diagnosis, which may represent a clinically significant underlying pathology. Macrothrombocytopenia, the presence of large platelets in combination with thrombocytopenia, can be acquired or hereditary and indicative of a complex disorder. In this review, we discuss the interpretation of platelet count and volume measured by automated haematology analysers and highlight some important technical considerations relevant to the analysis of blood samples with macrothrombocytopenia. We review how large cohorts, such as the UK Biobank and INTERVAL studies, have enabled an accurate description of the distribution and co-variation of platelet parameters in adult populations. We discuss how genome-wide association studies have identified hundreds of genetic associations with platelet count and mean platelet volume, which in aggregate can explain large fractions of phenotypic variance, consistent with a complex genetic architecture and polygenic inheritance. Finally, we describe the large genetic diagnostic and discovery programmes, which, simultaneously to genome-wide association studies, have expanded the repertoire of genes and variants associated with extreme platelet phenotypes. These have advanced our understanding of the pathogenesis of hereditary macrothrombocytopenia and support a future clinical diagnostic strategy that utilises genotype alongside clinical and laboratory phenotype data.

Whole Genome Interpretation for a Family of Five.

Although best practices have emerged on how to analyse and interpret personal genomes, the utility of whole genome screening remains underdeveloped. A large amount of information can be gathered from various types of analyses via whole genome sequencing including pathogenicity screening, genetic risk scoring, fitness, nutrition, and pharmacogenomic analysis. We recognize different levels of confidence when assessing the validity of genetic markers and apply rigorous standards for evaluation of phenotype associations. We illustrate the application of this approach on a family of five. By applying analyses of whole genomes from different methodological perspectives, we are able to build a more comprehensive picture to assist decision making in preventative healthcare and well-being management. Our interpretation and reporting outputs provide input for a clinician to develop a healthcare plan for the individual, based on genetic and other healthcare data.

Bayesian Inference Associates Rare KDR Variants with Specific Phenotypes in Pulmonary Arterial Hypertension.

Background - Approximately 25% of patients with pulmonary arterial hypertension (PAH) have been found to harbor rare mutations in disease-causing genes. To identify missing heritability in PAH we integrated deep phenotyping with whole-genome sequencing data using Bayesian statistics. Methods - We analyzed 13,037 participants enrolled in the NIHR BioResource - Rare Diseases (NBR) study, of which 1,148 were recruited to the PAH domain. To test for genetic associations between genes and selected phenotypes of pulmonary hypertension (PH), we used the Bayesian rare-variant association method BeviMed. Results - Heterozygous, high impact, likely loss-of-function variants in the Kinase Insert Domain Receptor (KDR) gene were strongly associated with significantly reduced transfer coefficient for carbon monoxide (KCO, posterior probability (PP)=0.989) and older age at diagnosis (PP=0.912). We also provide evidence for familial segregation of a rare nonsense KDR variant with these phenotypes. On computed tomographic imaging of the lungs, a range of parenchymal abnormalities were observed in the five patients harboring these predicted deleterious variants in KDR. Four additional PAH cases with rare likely loss-of-function variants in KDR were independently identified in the US PAH Biobank cohort with similar phenotypic characteristics. Conclusions - The Bayesian inference approach allowed us to independently validate KDR, which encodes for the Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), as a novel PAH candidate gene. Furthermore, this approach specifically associated high impact likely loss-of-function variants in the genetically constrained gene with distinct phenotypes. These findings provide evidence for KDR being a clinically actionable PAH gene and further support the central role of the vascular endothelium in the pathobiology of PAH.

The Polygenic and Monogenic Basis of Blood Traits and Diseases.

Blood cells play essential roles in human health, underpinning physiological processes such as immunity, oxygen transport, and clotting, which when perturbed cause a significant global health burden. Here we integrate data from UK Biobank and a large-scale international collaborative effort, including data for 563,085 European ancestry participants, and discover 5,106 new genetic variants independently associated with 29 blood cell phenotypes covering a range of variation impacting hematopoiesis. We holistically characterize the genetic architecture of hematopoiesis, assess the relevance of the omnigenic model to blood cell phenotypes, delineate relevant hematopoietic cell states influenced by regulatory genetic variants and gene networks, identify novel splice-altering variants mediating the associations, and assess the polygenic prediction potential for blood traits and clinical disorders at the interface of complex and Mendelian genetics. These results show the power of large-scale blood cell trait GWAS to interrogate clinically meaningful variants across a wide allelic spectrum of human variation.

Development and validation of a universal blood donor genotyping platform: a multinational prospective study.

Each year, blood transfusions save millions of lives. However, under current blood-matching practices, sensitization to non-self-antigens is an unavoidable adverse side effect of transfusion. We describe a universal donor typing platform that could be adopted by blood services worldwide to facilitate a universal extended blood-matching policy and reduce sensitization rates. This DNA-based test is capable of simultaneously typing most clinically relevant red blood cell (RBC), human platelet (HPA), and human leukocyte (HLA) antigens. Validation was performed, using samples from 7927 European, 27 South Asian, 21 East Asian, and 9 African blood donors enrolled in 2 national biobanks. We illustrated the usefulness of the platform by analyzing antibody data from patients sensitized with multiple RBC alloantibodies. Genotyping results demonstrated concordance of 99.91%, 99.97%, and 99.03% with RBC, HPA, and HLA clinically validated typing results in 89 371, 3016, and 9289 comparisons, respectively. Genotyping increased the total number of antigen typing results available from 110 980 to >1 200 000. Dense donor typing allowed identification of 2 to 6 times more compatible donors to serve 3146 patients with multiple RBC alloantibodies, providing at least 1 match for 176 individuals for whom previously no blood could be found among the same donors. This genotyping technology is already being used to type thousands of donors taking part in national genotyping studies. Extraction of dense antigen-typing data from these cohorts provides blood supply organizations with the opportunity to implement a policy of genomics-based precision matching of blood.

Novel manifestations of immune dysregulation and granule defects in gray platelet syndrome.

Gray platelet syndrome (GPS) is a rare recessive disorder caused by biallelic variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet α-granules, splenomegaly, and bone marrow (BM) fibrosis. Due to the rarity of GPS, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathologic features, we performed a detailed clinical genotypic and phenotypic study of 47 patients with GPS and identified 32 new etiologic variants in NBEAL2. The GPS patient cohort exhibited known phenotypes, including macrothrombocytopenia, BM fibrosis, megakaryocyte emperipolesis of neutrophils, splenomegaly, and elevated serum vitamin B12 levels. Novel clinical phenotypes were also observed, including reduced leukocyte counts and increased presence of autoimmune disease and positive autoantibodies. There were widespread differences in the transcriptome and proteome of GPS platelets, neutrophils, monocytes, and CD4 lymphocytes. Proteins less abundant in these cells were enriched for constituents of granules, supporting a role for Nbeal2 in the function of these organelles across a wide range of blood cells. Proteomic analysis of GPS plasma showed increased levels of proteins associated with inflammation and immune response. One-quarter of plasma proteins increased in GPS are known to be synthesized outside of hematopoietic cells, predominantly in the liver. In summary, our data show that, in addition to the well-described platelet defects in GPS, there are immune defects. The abnormal immune cells may be the drivers of systemic abnormalities such as autoimmune disease.

Publisher Correction: Whole-genome sequencing of a sporadic primary immunodeficiency cohort.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.