Purnima Sundar

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.

Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire

Antibody repertoire diversity, potentially as high as 1011 unique molecules in a single individual, confounds characterization by conventional sequence analyses. In this study, we present a general method for assessing human antibody sequence diversity displayed on phage using massively parallel pyrosequencing, a novel application of Kabat column-labeled profile Hidden Markov Models, and translated complementarity determining region (CDR) capture-recapture analysis. Pyrosequencing of domain amplicon and RCA PCR products generated 1.5 × 106 reads, including more than 1.9 × 105 high quality, full-length sequences of antibody variable fragment (Fv) variable domains. Novel methods for germline and CDR classification and fine characterization of sequence diversity in the 6 CDRs are presented. Diverse germline contributions to the repertoire with random heavy and light chain pairing are observed. All germline families were found to be represented in 1.7 × 104 sequences obtained from repeated panning of the library. While the most variable CDR (CDR-H3) presents significant length and sequence variability, we find a substantial contribution to total diversity from somatically mutated germline encoded CDRs 1 and 2. Using a capture-recapture method, the total diversity of the antibody library obtained from a human donor Immunoglobulin M (IgM) pool was determined to be at least 3.5 × 1010. The results provide insights into the role of IgM diversification, display library construction, and productive germline usages in antibody libraries and the humoral repertoire.

B cell exchange across the blood-brain barrier in multiple sclerosis

In multiple sclerosis (MS) pathogenic B cells likely act on both sides of the blood-brain barrier (BBB). However, it is unclear whether antigen-experienced B cells are shared between the CNS and the peripheral blood (PB) compartments. We applied deep repertoire sequencing of IgG heavy chain variable region genes (IgG-VH) in paired cerebrospinal fluid and PB samples from patients with MS and other neurological diseases to identify related B cells that are common to both compartments. For the first time to our knowledge, we found that a restricted pool of clonally related B cells participated in robust bidirectional exchange across the BBB. Some clusters of related IgG-VH appeared to have undergone active diversification primarily in the CNS, while others have undergone active diversification in the periphery or in both compartments in parallel. B cells are strong candidates for autoimmune effector cells in MS, and these findings suggest that CNS-directed autoimmunity may be triggered and supported on both sides of the BBB. These data also provide a powerful approach to identify and monitor B cells in the PB that correspond to clonally amplified populations in the CNS in MS and other inflammatory states.

Naive antibody gene-segment frequencies are heritable and unaltered by chronic lymphocyte ablation.

A diverse antibody repertoire is essential for an effective adaptive immune response to novel molecular surfaces. Although past studies have observed common patterns of V-segment use, as well as variation in V-segment use between individuals, the relative contributions to variance from genetics, disease, age, and environment have remained unclear. Using high-throughput sequence analysis of monozygotic twins, we show that variation in naive VH and DH segment use is strongly determined by an individuals germ-line genetic background. The inherited segment-use profiles are resilient to differential environmental exposure, disease processes, and chronic lymphocyte depletion therapy. Signatures of the inherited profiles were observed in class switched germ-line use of each individual. However, despite heritable segment use, the rearranged complementarity-determining region-H3 repertoires remained highly specific to the individual. As it has been previously demonstrated that certain V-segments exhibit biased representation in autoimmunity, lymphoma, and viral infection, we anticipate our findings may provide a unique mechanism for stratifying individual risk profiles in specific diseases.

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.

Synthetic Antibodies Designed on Natural Sequence Landscapes

We present a method for synthetic antibody library generation that combines the use of high-throughput immune repertoire analysis and a novel synthetic technology. The library design recapitulates positional amino acid frequencies observed in natural antibody repertoires. V-segment diversity in four heavy (V(H)) and two kappa (V(κ)) germlines was introduced based on the analysis of somatically hypermutated donor-derived repertoires. Complementarity-determining region 3 length and amino acid designs were based on aggregate frequencies of all V(H) and V(κ) sequences in the data set. The designed libraries were constructed through an adaptation of a novel gene synthesis technology that enables precise positional control of amino acid composition and incorporation frequencies. High-throughput pyrosequencing was used to monitor the fidelity of construction and characterize genetic diversity in the final 3.6×10(10) transformants. The library exhibited Fab expression superior to currently reported synthetic approaches of equivalent diversity, with greater than 93% of clones observed to successfully display both a correctly folded heavy chain and a correctly folded light chain. Genetic diversity in the library was high, with 95% of 7.0×10(5) clones sequenced observed only once. The obtained library diversity explores a comparable sequence space as the donor-derived natural repertoire and, at the same time, is able to access novel recombined diversity due to lack of segmental linkage. The successful isolation of low- and subnanomolar-affinity antibodies against a diverse panel of receptors, growth factors, enzymes, antigens from infectious reagents, and peptides confirms the functional viability of the design strategy.

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.

A Systematic Review of Social Media Use to Discuss and View Deliberate Self-Harm Acts

Objective To conduct a systematic review of studies of social media platforms used by young people to discuss and view deliberate self-harm. Study Design 11 electronic databases were searched from January 2000 to January 2012 for primary research; in June 2014 an updated search of Medline was conducted. Grey literature sources were also searched. Search results were screened by two reviewers. Data were extracted by one reviewer and verified by another. Methodological quality was assessed using the Mixed Methods Appraisal Tool. Results Due to heterogeneity in study objectives and outcomes, results were not pooled; a narrative analysis is presented. 26 studies were included. Most were conducted in Canada or the UK (30.8% each), used qualitative designs (42.3%), and evaluated discussion forums (73.1%). Participants were most often aged 19–21 years (69.2%), female (mean 68.6%), and 19.2% had a documented history of depression. The social media platforms evaluated were commonly supportive and provided a sense of community among users. Support included suggestions for formal treatment, advice on stopping self-harming behavior, and encouragement. Harms included normalizing and accepting self-harming behavior; discussion of motivation or triggers, concealment, suicidal ideation or plans; and live depictions of self-harm acts. Conclusions Although this evidence is limited by its descriptive nature, studies identify beneficial and detrimental effects for young people using social media to discuss and view deliberate self-harm. The connections users make online may be valuable to explore for therapeutic benefit. Prospective, longitudinal investigations are needed to identify short- and long-term potential harms associated with use.

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.