Steven J. Pitts

Whole-genome sequence variation, population structure and demographic history of the Dutch population

Whole-genome sequencing enables complete characterization of genetic variation, but geographic clustering of rare alleles demands many diverse populations be studied. Here we describe the Genome of the Netherlands (GoNL) Project, in which we sequenced the whole genomes of 250 Dutch parent-offspring families and constructed a haplotype map of 20.4 million single-nucleotide variants and 1.2 million insertions and deletions. The intermediate coverage (∼13×) and trio design enabled extensive characterization of structural variation, including midsize events (30–500 bp) previously poorly catalogued and de novo mutations. We demonstrate that the quality of the haplotypes boosts imputation accuracy in independent samples, especially for lower frequency alleles. Population genetic analyses demonstrate fine-scale structure across the country and support multiple ancient migrations, consistent with historical changes in sea level and flooding. The GoNL Project illustrates how single-population whole-genome sequencing can provide detailed characterization of genetic variation and may guide the design of future population studies.

Immunoglobulin class-switched B cells form an active immune axis between CNS and periphery in multiple sclerosis

Ig class-switched B cells connect the periphery and CNS and support peripheral antigen-driven B cell activation in CNS autoimmunity. B Cells Flip the Switch for MS B cells in multiple sclerosis (MS) patients may mature outside the central nervous system (CNS). Two complementary studies report that B cells found outside the CNS—in peripheral blood and draining cervical lymph nodes (CLNs)—share antigen specificity with intrathecal B cell repertoires. In patients with MS, immune cells attack the CNS; however, it remains unclear whether these cells mature in the CNS or traffic to the CNS as mature cells. Using paired tissues and high-throughput sequencing, Stern et al. found that clonally expanded B cells are found in both the CNS and CLNs, but that founding members were more often found in the draining CLNs. Palanichamy et al. extend these findings by reporting a peripheral blood/CNS axis of mature B cells that have undergone class switch. These data support the therapeutic use of monoclonal antibodies that prevent lymphocytes from crossing the blood-brain barrier or induce peripheral B cell depletion in MS patients. In multiple sclerosis (MS), lymphocyte—in particular B cell—transit between the central nervous system (CNS) and periphery may contribute to the maintenance of active disease. Clonally related B cells exist in the cerebrospinal fluid (CSF) and peripheral blood (PB) of MS patients; however, it remains unclear which subpopulations of the highly diverse peripheral B cell compartment share antigen specificity with intrathecal B cell repertoires and whether their antigen stimulation occurs on both sides of the blood-brain barrier. To address these questions, we combined flow cytometric sorting of PB B cell subsets with deep immune repertoire sequencing of CSF and PB B cells. Immunoglobulin (IgM and IgG) heavy chain variable (VH) region repertoires of five PB B cell subsets from MS patients were compared with their CSF Ig-VH transcriptomes. In six of eight patients, we identified peripheral CD27+IgD− memory B cells, CD27hiCD38hi plasma cells/plasmablasts, or CD27−IgD− B cells that had an immune connection to the CNS compartment. Pinpointing Ig class-switched B cells as key component of the immune axis thought to contribute to ongoing MS disease activity strengthens the rationale of current B cell–targeting therapeutic strategies and may lead to more targeted approaches.

Precise and Efficient Antibody Epitope Determination through Library Design, Yeast Display and Next-Generation Sequencing

The ability of antibodies to bind an antigen with a high degree of affinity and specificity has led them to become the largest and fastest growing class of therapeutic proteins. Clearly identifying the epitope at which they bind their cognate antigen provides insight into their mechanism of action and helps differentiate antibodies that bind the same antigen. Here, we describe a method to precisely and efficiently map the epitopes of a panel of antibodies in parallel over the course of several weeks. This method relies on the combination of rational library design, quantitative yeast surface display and next-generation DNA sequencing and was demonstrated by mapping the epitopes of several antibodies that neutralize alpha toxin from Staphylococcus aureus. The accuracy of this method was confirmed by comparing the results to the co-crystal structure of one antibody and alpha toxin and was further refined by the inclusion of a lower-affinity variant of the antibody. In addition, this method produced quantitative insight into the epitope residues most critical for the antibody-antigen interaction and enabled the relative affinities of each antibody toward alpha toxin variants to be estimated. This affinity estimate serves as a predictor of neutralizing antibody potency and was used to anticipate the ability of each antibody to effectively bind and neutralize naturally occurring alpha toxin variants secreted by strains of S. aureus, including clinically relevant strains. Ultimately this type information can be used to help select the best clinical candidate among a set of antibodies against a given antigen.

Aging as Accelerated Accumulation of Somatic Variants : Whole-Genome Sequencing of Centenarian and Middle-Aged Monozygotic Twin Pairs

It has been postulated that aging is the consequence of an accelerated accumulation of somatic DNA mutations and that subsequent errors in the primary structure of proteins ultimately reach levels sufficient to affect organismal functions. The technical limitations of detecting somatic changes and the lack of insight about the minimum level of erroneous proteins to cause an error catastrophe hampered any firm conclusions on these theories. In this study, we sequenced the whole genome of DNA in whole blood of two pairs of monozygotic (MZ) twins, 40 and 100 years old, by two independent next-generation sequencing (NGS) platforms (Illumina and Complete Genomics). Potentially discordant single-base substitutions supported by both platforms were validated extensively by Sanger, Roche 454, and Ion Torrent sequencing. We demonstrate that the genomes of the two twin pairs are germ-line identical between co-twins, and that the genomes of the 100-year-old MZ twins are discerned by eight confirmed somatic single-base substitutions, five of which are within introns. Putative somatic variation between the 40-year-old twins was not confirmed in the validation phase. We conclude from this systematic effort that by using two independent NGS platforms, somatic single nucleotide substitutions can be detected, and that a century of life did not result in a large number of detectable somatic mutations in blood. The low number of somatic variants observed by using two NGS platforms might provide a framework for detecting disease-related somatic variants in phenotypically discordant MZ twins.

Identification of novel risk loci for restless legs syndrome in genome-wide association studies in individuals of European ancestry: a meta-analysis

Summary Background Restless legs syndrome is a prevalent chronic neurological disorder with potentially severe mental and physical health consequences. Clearer understanding of the underlying pathophysiology is needed to improve treatment options. We did a meta-analysis of genome-wide association studies (GWASs) to identify potential molecular targets. Methods In the discovery stage, we combined three GWAS datasets (EU-RLS GENE, INTERVAL, and 23andMe) with diagnosis data collected from 2003 to 2017, in face-to-face interviews or via questionnaires, and involving 15 126 cases and 95 725 controls of European ancestry. We identified common variants by fixed-effect inverse-variance meta-analysis. Significant genome-wide signals (p≤5 × 10−8) were tested for replication in an independent GWAS of 30 770 cases and 286 913 controls, followed by a joint analysis of the discovery and replication stages. We did gene annotation, pathway, and gene-set-enrichment analyses and studied the genetic correlations between restless legs syndrome and traits of interest. Findings We identified and replicated 13 new risk loci for restless legs syndrome and confirmed the previously identified six risk loci. MEIS1 was confirmed as the strongest genetic risk factor for restless legs syndrome (odds ratio 1·92, 95% CI 1·85–1·99). Gene prioritisation, enrichment, and genetic correlation analyses showed that identified pathways were related to neurodevelopment and highlighted genes linked to axon guidance (associated with SEMA6D), synapse formation (NTNG1), and neuronal specification (HOXB cluster family and MYT1). Interpretation Identification of new candidate genes and associated pathways will inform future functional research. Advances in understanding of the molecular mechanisms that underlie restless legs syndrome could lead to new treatment options. We focused on common variants; thus, additional studies are needed to dissect the roles of rare and structural variations. Funding Deutsche Forschungsgemeinschaft, Helmholtz Zentrum München–Deutsches Forschungszentrum für Gesundheit und Umwelt, National Research Institutions, NHS Blood and Transplant, National Institute for Health Research, British Heart Foundation, European Commission, European Research Council, National Institutes of Health, National Institute of Neurological Disorders and Stroke, NIH Research Cambridge Biomedical Research Centre, and UK Medical Research Council.

Uncompromised 10-year survival of oldest old carrying somatic mutations in DNMT3A and TET2.

To the editor: Recent large-scale sequencing studies report recurrent somatic mutations in the blood of elderly individuals in genes previously linked to clonal expansion of hematopoietic stem cells.[1][1][⇓][2][⇓][3]-[4][4] Particularly for DNMT3A and TET2 , a steep age-associated increase in

Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire.

Staphylococcus aureus is both an important pathogen and a human commensal. To explore this ambivalent relationship between host and microbe, we analysed the memory humoral response against IsdB, a protein involved in iron acquisition, in four healthy donors. Here we show that in all donors a heavily biased use of two immunoglobulin heavy chain germlines generated high affinity (pM) antibodies that neutralize the two IsdB NEAT domains, IGHV4-39 for NEAT1 and IGHV1-69 for NEAT2. In contrast to the typical antibody/antigen interactions, the binding is primarily driven by the germline-encoded hydrophobic CDRH-2 motifs of IGHV1-69 and IGHV4-39, with a binding mechanism nearly identical for each antibody derived from different donors. Our results suggest that IGHV1-69 and IGHV4-39, while part of the adaptive immune system, may have evolved under selection pressure to encode a binding motif innately capable of recognizing and neutralizing a structurally conserved protein domain involved in pathogen iron acquisition.

A framework for the detection of de novo mutations in family-based sequencing data

Germline mutation detection from human DNA sequence data is challenging due to the rarity of such events relative to the intrinsic error rates of sequencing technologies and the uneven coverage across the genome. We developed PhaseByTransmission (PBT) to identify de novo single nucleotide variants and short insertions and deletions (indels) from sequence data collected in parent-offspring trios. We compute the joint probability of the data given the genotype likelihoods in the individual family members, the known familial relationships and a prior probability for the mutation rate. Candidate de novo mutations (DNMs) are reported along with their posterior probability, providing a systematic way to prioritize them for validation. Our tool is integrated in the Genome Analysis Toolkit and can be used together with the ReadBackedPhasing module to infer the parental origin of DNMs based on phase-informative reads. Using simulated data, we show that PBT outperforms existing tools, especially in low coverage data and on the X chromosome. We further show that PBT displays high validation rates on empirical parent-offspring sequencing data for whole-exome data from 104 trios and X-chromosome data from 249 parent-offspring families. Finally, we demonstrate an association between father’s age at conception and the number of DNMs in female offspring’s X chromosome, consistent with previous literature reports.

Effect of genome and environment on metabolic and inflammatory profiles.

Twin and family studies have established the contribution of genetic factors to variation in metabolic, hematologic and immunological parameters. The majority of these studies analyzed single or combined traits into pre-defined syndromes. In the present study, we explore an alternative multivariate approach in which a broad range of metabolic, hematologic, and immunological traits are analyzed simultaneously to determine the resemblance of monozygotic (MZ) twin pairs, twin-spouse pairs and unrelated, non-cohabiting individuals. A total of 517 participants from the Netherlands Twin Register, including 210 MZ twin pairs and 64 twin-spouse pairs, took part in the study. Data were collected on body composition, blood pressure, heart rate, and multiple biomarkers assessed in fasting blood samples, including lipid levels, glucose, insulin, liver enzymes, hematological measurements and cytokine levels. For all 51 measured traits, pair-wise Pearson correlations, correcting for family relatedness, were calculated across all the individuals in the cohort. Hierarchical clustering techniques were applied to group the measured traits into sub-clusters based on similarity. Sub-clusters were observed among metabolic traits and among inflammatory markers. We defined a phenotypic profile as the collection of all the traits measured for a given individual. Average within-pair similarity of phenotypic profiles was determined for the groups of MZ twin pairs, spouse pairs and pairs of unrelated individuals. The average similarity across the full phenotypic profile was higher for MZ twin pairs than for spouse pairs, and lowest for pairs of unrelated individuals. Cohabiting MZ twins were more similar in their phenotypic profile compared to MZ twins who no longer lived together. The correspondence in the phenotypic profile is therefore determined to a large degree by familial, mostly genetic, factors, while household factors contribute to a lesser degree to profile similarity.

Productive common light chain libraries yield diverse panels of high affinity bispecific antibodies

ABSTRACT The commercial success of bispecific antibodies generally has been hindered by the complexities associated with generating appropriate molecules for both research scale and large scale manufacturing purposes. Bispecific IgG (BsIgG) based on two antibodies that use an identical common light chain can be combined with a minimal set of Fc mutations to drive heavy chain heterodimerization in order to address these challenges. However, the facile generation of common light chain antibodies with properties similar to traditional monoclonal antibodies has not been demonstrated and they have only been used sparingly. Here, we describe the design of a synthetic human antibody library based on common light chains to generate antibodies with biochemical and biophysical properties that are indistinguishable to traditional therapeutic monoclonal antibodies. We used this library to generate diverse panels of well-behaved, high affinity antibodies toward a variety of epitopes across multiple antigens, including mouse 4-1BB, a therapeutically important T cell costimulatory receptor. Over 200 BsIgG toward 4-1BB were generated using an automated purification method we developed that enables milligram-scale production of BsIgG. This approach allowed us to identify antibodies with a wide range of agonistic activity that are being used to further investigate the therapeutic potential of antibodies targeting one or more epitopes of 4-1BB.