The landscape of antibody binding affinity in SARS-CoV-2 Omicron BA.1 evolution.

The Omicron BA.1 variant of SARS-CoV-2 escapes convalescent sera and monoclonal antibodies that are effective against earlier strains of the virus. This immune evasion is largely a consequence of mutations in the BA.1 receptor binding domain (RBD), the major antigenic target of SARS-CoV-2. Previous studies have identified several key RBD mutations leading to escape from most antibodies. However, little is known about how these escape mutations interact with each other and with other mutations in the RBD. Here, we systematically map these interactions by measuring the binding affinity of all possible combinations of these 15 RBD mutations (215=32,768 genotypes) to 4 monoclonal antibodies (LY-CoV016, LY-CoV555, REGN10987, and S309) with distinct epitopes. We find that BA.1 can lose affinity to diverse antibodies by acquiring a few large-effect mutations and can reduce affinity to others through several small-effect mutations. However, our results also reveal alternative pathways to antibody escape that does not include every large-effect mutation. Moreover, epistatic interactions are shown to constrain affinity decline in S309 but only modestly shape the affinity landscapes of other antibodies. Together with previous work on the ACE2 affinity landscape, our results suggest that the escape of each antibody is mediated by distinct groups of mutations, whose deleterious effects on ACE2 affinity are compensated by another distinct group of mutations (most notably Q498R and N501Y).

A pseudovirus system enables deep mutational scanning of the full SARS-CoV-2 spike.

A major challenge in understanding SARS-CoV-2 evolution is interpreting the antigenic and functional effects of emerging mutations in the viral spike protein. Here, we describe a deep mutational scanning platform based on non-replicative pseudotyped lentiviruses that directly quantifies how large numbers of spike mutations impact antibody neutralization and pseudovirus infection. We apply this platform to produce libraries of the Omicron BA.1 and Delta spikes. These libraries each contain ∼7,000 distinct amino acid mutations in the context of up to ∼135,000 unique mutation combinations. We use these libraries to map escape mutations from neutralizing antibodies targeting the receptor-binding domain, N-terminal domain, and S2 subunit of spike. Overall, this work establishes a high-throughput and safe approach to measure how ∼105 combinations of mutations affect antibody neutralization and spike-mediated infection. Notably, the platform described here can be extended to the entry proteins of many other viruses.

Decoding the effects of spike receptor binding domain mutations on antibody escape abilities of omicron variants.

Recent times witnessed an upsurge in the number of COVID19 cases which is primarily attributed to the emergence of several omicron variants, although there is substantial population vaccination coverage across the globe. Currently, many therapeutic antibodies have been approved for emergency usage. The present study critically evaluates the effect of mutations observed in several omicron variants on the binding affinities of different classes of RBD-specific antibodies using a combined approach of immunoinformatics and binding free energy calculations. Our binding affinity data clearly show that omicron variants achieve antibody escape abilities by incorporating mutations at the immunogenic hotspot residues for each specific class of antibody. K417N and Y505H point mutations are primarily accountable for the loss of class I antibody binding affinities. The K417N/Q493R/Q498R/Y505H combined mutant significantly reduces binding affinities for all the class I antibodies. E484A single mutation, on the other hand, drastically reduces binding affinities for most of the class II antibodies. E484A and E484A/Q493R double mutations cause a 33-38% reduction in binding affinity for an approved therapeutic monoclonal antibody. The Q498R RBD mutation observed across all the omicron variants can reduce ∼12% binding affinity for REGN10987, a class III therapeutic antibody, and the L452R/Q498R double mutation causes a ∼6% decrease in binding affinities for another class III therapeutic antibody, LY-CoV1404. Our data suggest that achieving the immune evasion abilities appears to be the selection pressure behind the emergence of omicron variants.

Molecular mechanisms responsible for SARS-CoV-2 antibody waning and vaccine escape in Omicron sublineages BA.4 and BA.5.

Mutations in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome continue to threaten the global landscape of the coronavirus disease 2019 (COVID-19) pandemic. The Omicron variant (B.1.1.529) rapidly displaced previous 'variants of concern' (VoC) in 2021 due to its high rate of transmissibility and multitude of mutations. This global influx of infections saturated healthcare systems, overwhelmed testing capacity and case reporting, and increased the COVID-19 death toll. Global health leaders are now being faced with the most transmissible COVID-19 variants yet, the Omicron sublineages BA.4 and BA.5, which contain additional spike protein (S) mutations from previous Omicron and VoC serotypes. With universally observed antibody waning, increasing vaccine-variant mismatch, and resuming international travel, the stage is set for unprecedented levels of breakthrough infections and superspreading events. In this paper, we raise awareness to these novel variants and provide context for the high likelihood of an upcoming wave of infection capable of inflicting significant disease burden on a global scale.

Integrating Conformational Dynamics and Perturbation-Based Network Modeling for Mutational Profiling of Binding and Allostery in the SARS-CoV-2 Spike Variant Complexes with Antibodies: Balancing Local and Global Determinants of Mutational Escape Mechanisms.

In this study, we combined all-atom MD simulations, the ensemble-based mutational scanning of protein stability and binding, and perturbation-based network profiling of allosteric interactions in the SARS-CoV-2 spike complexes with a panel of cross-reactive and ultra-potent single antibodies (B1-182.1 and A23-58.1) as well as antibody combinations (A19-61.1/B1-182.1 and A19-46.1/B1-182.1). Using this approach, we quantify the local and global effects of mutations in the complexes, identify protein stability centers, characterize binding energy hotspots, and predict the allosteric control points of long-range interactions and communications. Conformational dynamics and distance fluctuation analysis revealed the antibody-specific signatures of protein stability and flexibility of the spike complexes that can affect the pattern of mutational escape. A network-based perturbation approach for mutational profiling of allosteric residue potentials revealed how antibody binding can modulate allosteric interactions and identified allosteric control points that can form vulnerable sites for mutational escape. The results show that the protein stability and binding energetics of the SARS-CoV-2 spike complexes with the panel of ultrapotent antibodies are tolerant to the effect of Omicron mutations, which may be related to their neutralization efficiency. By employing an integrated analysis of conformational dynamics, binding energetics, and allosteric interactions, we found that the antibodies that neutralize the Omicron spike variant mediate the dominant binding energy hotpots in the conserved stability centers and allosteric control points in which mutations may be restricted by the requirements of the protein folding stability and binding to the host receptor. This study suggested a mechanism in which the patterns of escape mutants for the ultrapotent antibodies may not be solely determined by the binding interaction changes but are associated with the balance and tradeoffs of multiple local and global factors, including protein stability, binding affinity, and long-range interactions.

Structural Plasticity and Immune Evasion of SARS-CoV-2 Spike Variants.

The global pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has significantly affected every human life and overloaded the health care system worldwide. Limited therapeutic options combined with the consecutive waves of the infection and emergence of novel SARS-CoV-2 variants, especially variants of concern (VOCs), have prolonged the COVID-19 pandemic and challenged its control. The Spike (S) protein on the surface of SARS-CoV-2 is the primary target exposed to the host and essential for virus entry into cells. The parental (Wuhan-Hu-1 or USA/WA1 strain) S protein is the virus-specific component of currently implemented vaccines. However, S is most prone to mutations, potentially shifting the dynamics of virus-host interactions by affecting S conformational/structural profiles. Scientists have rapidly resolved atomic structures of S VOCs and elucidated molecular details of these mutations, which can inform the design of S-directed novel therapeutics and broadly protective vaccines. Here, we discuss recent findings on S-associated virus transmissibility and immune evasion of SARS-CoV-2 VOCs and experimental approaches used to profile these properties. We summarize the structural studies that document the structural flexibility/plasticity of S VOCs and the potential roles of accumulated mutations on S structures and functions. We focus on the molecular interpretation of structures of the S variants and its insights into the molecular mechanism underlying antibody evasion and host cell-receptor binding.

Antibody escape of SARS-CoV-2 Omicron BA.4 and BA.5 from vaccine and BA.1 serum.

The Omicron lineage of SARS-CoV-2, which was first described in November 2021, spread rapidly to become globally dominant and has split into a number of sublineages. BA.1 dominated the initial wave but has been replaced by BA.2 in many countries. Recent sequencing from South Africa's Gauteng region uncovered two new sublineages, BA.4 and BA.5, which are taking over locally, driving a new wave. BA.4 and BA.5 contain identical spike sequences, and although closely related to BA.2, they contain further mutations in the receptor-binding domain of their spikes. Here, we study the neutralization of BA.4/5 using a range of vaccine and naturally immune serum and panels of monoclonal antibodies. BA.4/5 shows reduced neutralization by the serum from individuals vaccinated with triple doses of AstraZeneca or Pfizer vaccine compared with BA.1 and BA.2. Furthermore, using the serum from BA.1 vaccine breakthrough infections, there are, likewise, significant reductions in the neutralization of BA.4/5, raising the possibility of repeat Omicron infections.

Tracking SARS-CoV-2 mutations and variants through the COG-UK-Mutation Explorer.

COG-UK Mutation Explorer (COG-UK-ME, https://sars2.cvr.gla.ac.uk/cog-uk/-last accessed date 16 March 2022) is a web resource that displays knowledge and analyses on SARS-CoV-2 virus genome mutations and variants circulating in the UK, with a focus on the observed amino acid replacements that have an antigenic role in the context of the human humoral and cellular immune response. This analysis is based on more than 2 million genome sequences (as of March 2022) for UK SARS-CoV-2 data held in the CLIMB-COVID centralised data environment. COG-UK-ME curates these data and displays analyses that are cross-referenced to experimental data collated from the primary literature. The aim is to track mutations of immunological importance that are accumulating in current variants of concern and variants of interest that could alter the neutralising activity of monoclonal antibodies (mAbs), convalescent sera, and vaccines. Changes in epitopes recognised by T cells, including those where reduced T cell binding has been demonstrated, are reported. Mutations that have been shown to confer SARS-CoV-2 resistance to antiviral drugs are also included. Using visualisation tools, COG-UK-ME also allows users to identify the emergence of variants carrying mutations that could decrease the neutralising activity of both mAbs present in therapeutic cocktails, e.g. Ronapreve. COG-UK-ME tracks changes in the frequency of combinations of mutations and brings together the curated literature on the impact of those mutations on various functional aspects of the virus and therapeutics. Given the unpredictable nature of SARS-CoV-2 as exemplified by yet another variant of concern, Omicron, continued surveillance of SARS-CoV-2 remains imperative to monitor virus evolution linked to the efficacy of therapeutics.

Emergence and onward transmission of a SARS-CoV-2 E484K variant among household contacts of a bamlanivimab-treated patient.

The implementation of monoclonal antibody therapeutics during the COVID-19 pandemic altered the selective pressures encountered by SARS-CoV-2, raising the possibility of selection for resistant variants. Within-host viral evolution was reported in treated immunocompromised individuals but whether this signifies a real risk of onward transmission is unclear. We used a regional SARS-CoV-2 sequencing program to monitor lineages with clinically relevant variants in identified patients, which facilitated analysis of parameters potentially relevant to new variant emergence. Here we describe a newly acquired spike E484K mutation detected within the B.1.311 lineage. Multiple individuals in 2 households of the same extended family were infected. The timing and patterns of spread were consistent with de novo emergence of this E484K variant in the bamlanivimab-treated index patient. Our study suggests that the selective pressures introduced by the widespread administration of these antibodies may warrant increased genomic surveillance to identify and mitigate spread of therapy-induced variants.

Insights on the mutational landscape of the SARS-CoV-2 Omicron variant receptor-binding domain.

The Omicron variant features enhanced transmissibility and antibody escape. Here, we describe the Omicron receptor-binding domain (RBD) mutational landscape using amino acid interaction (AAI) networks, which are well suited for interrogating constellations of mutations that function in an epistatic manner. Using AAI, we map Omicron mutations directly and indirectly driving increased escape breadth and depth in class 1-4 antibody epitopes. Further, we present epitope networks for authorized therapeutic antibodies and assess perturbations to each antibody's epitope. Since our initial modeling following the identification of Omicron, these predictions have been realized by experimental findings of Omicron neutralization escape from therapeutic antibodies ADG20, AZD8895, and AZD1061. Importantly, the AAI predicted escape resulting from indirect epitope perturbations was not captured by previous sequence or point mutation analyses. Finally, for several Omicron RBD mutations, we find evidence for a plausible role in enhanced transmissibility via disruption of RBD-down conformational stability at the RBDdown-RBDdown interface.

SARS-CoV-2 Variant Exposures Elicit Antibody Responses With Differential Cross-Neutralization of Established and Emerging Strains Including Delta and Omicron.

The wide spectrum of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with phenotypes impacting transmission and antibody sensitivity necessitates investigation of immune responses to different spike protein versions. Here, we compare neutralization of variants of concern, including B.1.617.2 (delta) and B.1.1.529 (omicron), in sera from individuals exposed to variant infection, vaccination, or both. We demonstrate that neutralizing antibody responses are strongest against variants sharing certain spike mutations with the immunizing exposure, and exposure to multiple spike variants increases breadth of variant cross-neutralization. These findings contribute to understanding relationships between exposures and antibody responses and may inform booster vaccination strategies.

RBD Double Mutations of SARS-CoV-2 Strains Increase Transmissibility through Enhanced Interaction between RBD and ACE2 Receptor.

The COVID-19 pandemic, caused by SARS-CoV-2, has led to catastrophic damage for global human health. The initial step of SARS-CoV-2 infection is the binding of the receptor-binding domain (RBD) in its spike protein to the ACE2 receptor in the host cell membrane. Constant evolution of SARS-CoV-2 generates new mutations across its genome including the coding region for the RBD in the spike protein. In addition to the well-known single mutation in the RBD, the recent new mutation strains with an RBD "double mutation" are causing new outbreaks globally, as represented by the delta strain containing RBD L452R/T478K. Although it is considered that the increased transmissibility of double-mutated strains could be attributed to the altered interaction between the RBD and ACE2 receptor, the molecular details remain to be elucidated. Using the methods of molecular dynamics simulation, superimposed structural comparison, free binding energy estimation, and antibody escaping, we investigated the relationship between the ACE2 receptor and the RBD double mutants of L452R/T478K (delta), L452R/E484Q (kappa), and E484K/N501Y (beta, gamma). The results demonstrated that each of the three RBD double mutants altered the RBD structure and enhanced the binding of the mutated RBD to ACE2 receptor. Together with the mutations in other parts of the virus genome, the double mutations increase the transmissibility of SARS-CoV-2 to host cells.

Structure and Mutations of SARS-CoV-2 Spike Protein: A Focused Overview.

The spike protein (S-protein) of SARS-CoV-2, the protein that enables the virus to infect human cells, is the basis for many vaccines and a hotspot of concerning virus evolution. Here, we discuss the outstanding progress in structural characterization of the S-protein and how these structures facilitate analysis of virus function and evolution. We emphasize the differences in reported structures and that analysis of structure-function relationships is sensitive to the structure used. We show that the average residue solvent exposure in nearly complete structures is a good descriptor of open vs closed conformation states. Because of structural heterogeneity of functionally important surface-exposed residues, we recommend using averages of a group of high-quality protein structures rather than a single structure before reaching conclusions on specific structure-function relationships. To illustrate these points, we analyze some significant chemical tendencies of prominent S-protein mutations in the context of the available structures. In the discussion of new variants, we emphasize the selectivity of binding to ACE2 vs prominent antibodies rather than simply the antibody escape or ACE2 affinity separately. We note that larger chemical changes, in particular increased electrostatic charge or side-chain volume of exposed surface residues, are recurring in mutations of concern, plausibly related to adaptation to the negative surface potential of human ACE2. We also find indications that the fixated mutations of the S-protein in the main variants are less destabilizing than would be expected on average, possibly pointing toward a selection pressure on the S-protein. The richness of available structures for all of these situations provides an enormously valuable basis for future research into these structure-function relationships.

Evolutionary and Phenotypic Characterization of Two Spike Mutations in European Lineage 20E of SARS-CoV-2.

We have detected two mutations in the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at amino acid positions 1163 and 1167 that appeared independently in multiple transmission clusters and different genetic backgrounds. Furthermore, both mutations appeared together in a cluster of 1,627 sequences belonging to clade 20E. This cluster is characterized by 12 additional single nucleotide polymorphisms but no deletions. The available structural information on the S protein in the pre- and postfusion conformations predicts that both mutations confer rigidity, which could potentially decrease viral fitness. Accordingly, we observed reduced infectivity of this spike genotype relative to the ancestral 20E sequence in vitro, and the levels of viral RNA in nasopharyngeal swabs were not significantly higher. Furthermore, the mutations did not impact thermal stability or antibody neutralization by sera from vaccinated individuals but moderately reduce neutralization by convalescent-phase sera from the early stages of the pandemic. Despite multiple successful appearances of the two spike mutations during the first year of SARS-CoV-2 evolution, the genotype with both mutations was displaced upon the expansion of the 20I (Alpha) variant. The midterm fate of the genotype investigated was consistent with the lack of advantage observed in the clinical and experimental data. IMPORTANCE We observed repeated, independent emergence of mutations in the SARS-CoV-2 spike involving amino acids 1163 and 1167, within the HR2 functional motif. Conclusions derived from evolutionary and genomic diversity analysis suggest that the co-occurrence of both mutations might pose an advantage for the virus and therefore a threat to effective control of the epidemic. However, biological characterization, including in vitro experiments and analysis of clinical data, indicated no clear benefit in terms of stability or infectivity. In agreement with this, continuous epidemiological surveillance conducted months after the first observations revealed that both mutations did not successfully outcompete other variants and stopped circulating 9 months after their initial detection. Additionally, we evaluated the potential of both mutations to escape neutralizing antibodies, finding that the presence of these two mutations on their own is not likely to confer antibody escape. Our results provide an example of how newly emerged spike mutations can be assessed to better understand the risk posed by new variants and indicate that some spike mutations confer no clear advantage to the virus despite independently emerging multiple times and are eventually displaced by fitter variants.

SARS-CoV-2 B.1.617 Mutations L452R and E484Q Are Not Synergistic for Antibody Evasion.

The SARS-CoV-2 B.1.617 variant emerged in the Indian state of Maharashtra in late 2020. There have been fears that 2 key mutations seen in the receptor-binding domain, L452R and E484Q, would have additive effects on evasion of neutralizing antibodies. We report that spike bearing L452R and E484Q confers modestly reduced sensitivity to BNT162b2 mRNA vaccine-elicited antibodies following either first or second dose. The effect is similar in magnitude to the loss of sensitivity conferred by L452R or E484Q alone. These data demonstrate reduced sensitivity to vaccine-elicited neutralizing antibodies by L452R and E484Q but lack of synergistic loss of sensitivity.

The ongoing evolution of variants of concern and interest of SARS-CoV-2 in Brazil revealed by convergent indels in the amino (N)-terminal domain of the spike protein.

Mutations at both the receptor-binding domain (RBD) and the amino (N)-terminal domain (NTD) of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spike (S) glycoprotein can alter its antigenicity and promote immune escape. We identified that SARS-CoV-2 lineages circulating in Brazil with mutations of concern in the RBD independently acquired convergent deletions and insertions in the NTD of the S protein, which altered the NTD antigenic-supersite and other predicted epitopes at this region. Importantly, we detected the community transmission of different P.1 lineages bearing NTD indels ∆69-70 (which can impact several SARS-CoV-2 diagnostic protocols), ∆144 and ins214ANRN, and a new VOI N.10 derived from the B.1.1.33 lineage carrying three NTD deletions (∆141-144, ∆211, and ∆256-258). These findings support that the ongoing widespread transmission of SARS-CoV-2 in Brazil generates new viral lineages that might be more resistant to antibody neutralization than parental variants of concern.

Complete Mapping of Mutations to the SARS-CoV-2 Spike Receptor-Binding Domain that Escape Antibody Recognition.

Antibodies targeting the SARS-CoV-2 spike receptor-binding domain (RBD) are being developed as therapeutics and are a major contributor to neutralizing antibody responses elicited by infection. Here, we describe a deep mutational scanning method to map how all amino-acid mutations in the RBD affect antibody binding and apply this method to 10 human monoclonal antibodies. The escape mutations cluster on several surfaces of the RBD that broadly correspond to structurally defined antibody epitopes. However, even antibodies targeting the same surface often have distinct escape mutations. The complete escape maps predict which mutations are selected during viral growth in the presence of single antibodies. They further enable the design of escape-resistant antibody cocktails-including cocktails of antibodies that compete for binding to the same RBD surface but have different escape mutations. Therefore, complete escape-mutation maps enable rational design of antibody therapeutics and assessment of the antigenic consequences of viral evolution.

Mechanisms of Hepatitis C Virus Escape from Vaccine-Relevant Neutralizing Antibodies.

Hepatitis C virus (HCV) is a major causative agent of acute and chronic hepatitis. It is estimated that 400,000 people die every year from chronic HCV infection, mostly from severe liver-related diseases such as cirrhosis and liver cancer. Although HCV was discovered more than 30 years ago, an efficient prophylactic vaccine is still missing. The HCV glycoprotein complex, E1/E2, is the principal target of neutralizing antibodies (NAbs) and, thus, is an attractive antigen for B-cell vaccine design. However, the high genetic variability of the virus necessitates the identification of conserved epitopes. Moreover, the high intrinsic mutational capacity of HCV allows the virus to continually escape broadly NAbs (bNAbs), which is likely to cause issues with vaccine-resistant variants. Several studies have assessed the barrier-to-resistance of vaccine-relevant bNAbs in vivo and in vitro. Interestingly, recent studies have suggested that escape substitutions can confer antibody resistance not only by direct modification of the epitope but indirectly through allosteric effects, which can be grouped based on the breadth of these effects on antibody susceptibility. In this review, we summarize the current understanding of HCV-specific NAbs, with a special focus on vaccine-relevant bNAbs and their targets. We highlight antibody escape studies pointing out the different methodologies and the escape mutations identified thus far. Finally, we analyze the antibody escape mechanisms of envelope protein escape substitutions and polymorphisms according to the most recent evidence in the HCV field. The accumulated knowledge in identifying bNAb epitopes as well as assessing barriers to resistance and elucidating relevant escape mechanisms may prove critical in the successful development of an HCV B-cell vaccine.

Complete map of SARS-CoV-2 RBD mutations that escape the monoclonal antibody LY-CoV555 and its cocktail with LY-CoV016.

Monoclonal antibodies and antibody cocktails are a promising therapeutic and prophylaxis for coronavirus disease 2019 (COVID-19). However, ongoing evolution of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) can render monoclonal antibodies ineffective. Here, we completely map all of the mutations to the SARS-CoV-2 spike receptor-binding domain (RBD) that escape binding by a leading monoclonal antibody, LY-CoV555, and its cocktail combination with LY-CoV016. Individual mutations that escape binding by each antibody are combined in the circulating B.1.351 and P.1 SARS-CoV-2 lineages (E484K escapes LY-CoV555, K417N/T escapes LY-CoV016). In addition, the L452R mutation in the B.1.429 lineage escapes LY-CoV555. Furthermore, we identify single amino acid changes that escape the combined LY-CoV555+LY-CoV016 cocktail. We suggest that future efforts diversify the epitopes targeted by antibodies and antibody cocktails to make them more resilient to the antigenic evolution of SARS-CoV-2.

Recurrent emergence of SARS-CoV-2 spike deletion H69/V70 and its role in the Alpha variant B.1.1.7.

We report severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike ΔH69/V70 in multiple independent lineages, often occurring after acquisition of receptor binding motif replacements such as N439K and Y453F, known to increase binding affinity to the ACE2 receptor and confer antibody escape. In vitro, we show that, although ΔH69/V70 itself is not an antibody evasion mechanism, it increases infectivity associated with enhanced incorporation of cleaved spike into virions. ΔH69/V70 is able to partially rescue infectivity of spike proteins that have acquired N439K and Y453F escape mutations by increased spike incorporation. In addition, replacement of the H69 and V70 residues in the Alpha variant B.1.1.7 spike (where ΔH69/V70 occurs naturally) impairs spike incorporation and entry efficiency of the B.1.1.7 spike pseudotyped virus. Alpha variant B.1.1.7 spike mediates faster kinetics of cell-cell fusion than wild-type Wuhan-1 D614G, dependent on ΔH69/V70. Therefore, as ΔH69/V70 compensates for immune escape mutations that impair infectivity, continued surveillance for deletions with functional effects is warranted.