Sarah A. Graham

Decoding the genetics of speech and language

Researchers are beginning to uncover the neurogenetic pathways that underlie our unparalleled capacity for spoken language. Initial clues come from identification of genetic risk factors implicated in developmental language disorders. The underlying genetic architecture is complex, involving a range of molecular mechanisms. For example, rare protein-coding mutations of the FOXP2 transcription factor cause severe problems with sequencing of speech sounds, while common genetic risk variants of small effect size in genes like CNTNAP2, ATP2C2 and CMIP are associated with typical forms of language impairment. In this article, we describe how investigations of these and other candidate genes, in humans, animals and cellular models, are unravelling the connections between genes and cognition. This depends on interdisciplinary research at multiple levels, from determining molecular interactions and functional roles in neural cell-biology all the way through to effects on brain structure and activity.

Structural basis for langerin recognition of diverse pathogen and mammalian glycans through a single binding site.

Langerin mediates the carbohydrate-dependent uptake of pathogens by Langerhans cells in the first step of antigen presentation to the adaptive immune system. Langerin binds to an unusually diverse number of endogenous and pathogenic cell surface carbohydrates, including mannose-containing O-specific polysaccharides derived from bacterial lipopolysaccharides identified here by probing a microarray of bacterial polysaccharides. Crystal structures of the carbohydrate-recognition domain from human langerin bound to a series of oligomannose compounds, the blood group B antigen, and a fragment of β-glucan reveal binding to mannose, fucose, and glucose residues by Ca2+ coordination of vicinal hydroxyl groups with similar stereochemistry. Oligomannose compounds bind through a single mannose residue, with no other mannose residues contacting the protein directly. There is no evidence for a second Ca2+-independent binding site. Likewise, a β-glucan fragment, Glcβ1–3Glcβ1–3Glc, binds to langerin through the interaction of a single glucose residue with the Ca2+ site. The fucose moiety of the blood group B trisaccharide Galα1–3(Fucα1–2)Gal also binds to the Ca2+ site, and selective binding to this glycan compared to other fucose-containing oligosaccharides results from additional favorable interactions of the nonreducing terminal galactose, as well as of the fucose residue. Surprisingly, the equatorial 3-OH group and the axial 4-OH group of the galactose residue in 6SO4–Galβ1–4GlcNAc also coordinate Ca2+, a heretofore unobserved mode of galactose binding in a C-type carbohydrate-recognition domain bearing the Glu-Pro-Asn signature motif characteristic of mannose binding sites. Salt bridges between the sulfate group and two lysine residues appear to compensate for the nonoptimal binding of galactose at this site.

Targeted glycoproteomic identification of cancer cell glycosylation

GalMBP is a fragment of serum mannose-binding protein that has been modified to create a probe for galactose-containing ligands. Glycan array screening demonstrated that the carbohydrate-recognition domain of GalMBP selectively binds common groups of tumor-associated glycans, including Lewis-type structures and T antigen, suggesting that engineered glycan-binding proteins such as GalMBP represent novel tools for the characterization of glycoproteins bearing tumor-associated glycans. Blotting of cell extracts and membranes from MCF7 breast cancer cells with radiolabeled GalMBP was used to demonstrate that it binds to a selected set of high molecular weight glycoproteins that could be purified from MCF7 cells on an affinity column constructed with GalMBP. Proteomic and glycomic analysis of these glycoproteins by mass spectrometry showed that they are forms of CD98hc that bear glycans displaying heavily fucosylated termini, including Lewisx and Lewisy structures. The pool of ligands was found to include the target ligands for anti-CD15 antibodies, which are commonly used to detect Lewisx antigen on tumors, and for the endothelial scavenger receptor C-type lectin, which may be involved in tumor metastasis through interactions with this antigen. A survey of additional breast cancer cell lines reveals that there is wide variation in the types of glycosylation that lead to binding of GalMBP. Higher levels of binding are associated either with the presence of outer-arm fucosylated structures carried on a variety of different cell surface glycoproteins or with the presence of high levels of the mucin MUC1 bearing T antigen.

Selective Binding of the Scavenger Receptor C-type Lectin to Lewisx Trisaccharide and Related Glycan Ligands

The scavenger receptor C-type lectin (SRCL) is an endothelial receptor that is similar in organization to type A scavenger receptors for modified low density lipoproteins but contains a C-type carbohydrate-recognition domain (CRD). Fragments of the receptor consisting of the entire extracellular domain and the CRD have been expressed and characterized. The extracellular domain is a trimer held together by collagen-like and coiled-coil domains adjacent to the CRD. The amino acid sequence of the CRD is very similar to the CRD of the asialoglycoprotein receptor and other galactose-specific receptors, but SRCL binds selectively to asialo-orosomucoid rather than generally to asialoglycoproteins. Screening of a glycan array and further quantitative binding studies indicate that this selectivity results from high affinity binding to glycans bearing the Lewisx trisaccharide. Thus, SRCL shares with the dendritic cell receptor DC-SIGN the ability to bind the Lewisx epitope. However, it does so in a fundamentally different way, making a primary binding interaction with the galactose moiety of the glycan rather than the fucose residue. SRCL shares with the asialoglycoprotein receptor the ability to mediate endocytosis and degradation of glycoprotein ligands. These studies suggest that SRCL might be involved in selective clearance of specific desialylated glycoproteins from circulation and/or interaction of cells bearing Lewisx-type structures with the vascular endothelium.

Binding-site geometry and flexibility in DC-SIGN demonstrated with surface force measurements

The dendritic cell receptor DC-SIGN mediates pathogen recognition by binding to glycans characteristic of pathogen surfaces, including those found on HIV. Clustering of carbohydrate-binding sites in the receptor tetramer is believed to be critical for targeting of pathogen glycans, but the arrangement of these sites remains poorly understood. Surface force measurements between apposed lipid bilayers displaying the extracellular domain of DC-SIGN and a neoglycolipid bearing an oligosaccharide ligand provide evidence that the receptor is in an extended conformation and that glycan docking is associated with a conformational change that repositions the carbohydrate-recognition domains during ligand binding. The results further show that the lateral mobility of membrane-bound ligands enhances the engagement of multiple carbohydrate-recognition domains in the receptor oligomer with appropriately spaced ligands. These studies highlight differences between pathogen targeting by DC-SIGN and receptors in which binding sites at fixed spacing bind to simple molecular patterns.

Understanding Language from a Genomic Perspective

Language is a defining characteristic of the human species, but its foundations remain mysterious. Heritable disorders offer a gateway into biological underpinnings, as illustrated by the discovery that FOXP2 disruptions cause a rare form of speech and language impairment. The genetic architecture underlying language-related disorders is complex, and although some progress has been made, it has proved challenging to pinpoint additional relevant genes with confidence. Next-generation sequencing and genome-wide association studies are revolutionizing understanding of the genetic bases of other neurodevelopmental disorders, like autism and schizophrenia, and providing fundamental insights into the molecular networks crucial for typical brain development. We discuss how a similar genomic perspective, brought to the investigation of language-related phenotypes, promises to yield equally informative discoveries. Moreover, we outline how follow-up studies of genetic findings using cellular systems and animal models can help to elucidate the biological mechanisms involved in the development of brain circuits supporting language.

Identification and functional characterization of de novo FOXP1 variants provides novel insights into the etiology of neurodevelopmental disorder

De novo disruptions of the neural transcription factor FOXP1 are a recently discovered, rare cause of sporadic intellectual disability (ID). We report three new cases of FOXP1-related disorder identified through clinical whole-exome sequencing. Detailed phenotypic assessment confirmed that global developmental delay, autistic features, speech/language deficits, hypotonia and mild dysmorphic features are core features of the disorder. We expand the phenotypic spectrum to include sensory integration disorder and hypertelorism. Notably, the etiological variants in these cases include two missense variants within the DNA-binding domain of FOXP1. Only one such variant has been reported previously. The third patient carries a stop-gain variant. We performed functional characterization of the three missense variants alongside our stop-gain and two previously described truncating/frameshift variants. All variants severely disrupted multiple aspects of protein function. Strikingly, the missense variants had similarly severe effects on protein function as the truncating/frameshift variants. Our findings indicate that a loss of transcriptional repression activity of FOXP1 underlies the neurodevelopmental phenotype in FOXP1-related disorder. Interestingly, the three novel variants retained the ability to interact with wild-type FOXP1, suggesting these variants could exert a dominant-negative effect by interfering with the normal FOXP1 protein. These variants also retained the ability to interact with FOXP2, a paralogous transcription factor disrupted in rare cases of speech and language disorder. Thus, speech/language deficits in these individuals might be worsened through deleterious effects on FOXP2 function. Our findings highlight that de novo FOXP1 variants are a cause of sporadic ID and emphasize the importance of this transcription factor in neurodevelopment.

Identification of Neutrophil Granule Glycoproteins as Lewisx-containing Ligands Cleared by the Scavenger Receptor C-type Lectin

The scavenger receptor C-type lectin (SRCL) is a glycan-binding receptor that has the capacity to mediate endocytosis of glycoproteins carrying terminal Lewisx groups (Galβ1–4(Fucα1–3)GlcNAc). A screen for glycoprotein ligands for SRCL using affinity chromatography on immobilized SRCL followed by mass spectrometry-based proteomic analysis revealed that soluble glycoproteins from secondary granules of neutrophils, including lactoferrin and matrix metalloproteinases 8 and 9, are major ligands. Binding competition and surface plasmon resonance analysis showed affinities in the low micromolar range. Comparison of SRCL binding to neutrophil and milk lactoferrin indicates that the binding is dependent on cell-specific glycosylation in the neutrophils, as the milk form of the glycoprotein is a much poorer ligand. Binding to neutrophil glycoproteins is fucose-dependent, and mass spectrometry-based glycomic analysis of neutrophil and milk lactoferrin was used to establish a correlation between high affinity binding to SRCL and the presence of multiple clustered terminal Lewisx groups on a heterogeneous mixture of branched glycans, some with poly N-acetyllactosamine extensions. The ability of SRCL to mediate uptake of neutrophil lactoferrin was confirmed using fibroblasts transfected with SRCL. The common presence of Lewisx groups in granule protein glycans can thus target granule proteins for clearance by SRCL. PCR and immunohistochemical analysis confirm that SRCL is widely expressed on endothelial cells and thus represents a distributed system that could scavenge released neutrophil glycoproteins both locally at sites of inflammation or systemically when they are released in the circulation.

BCL11A Haploinsufficiency Causes an Intellectual Disability Syndrome and Dysregulates Transcription

Intellectual disability (ID) is a common condition with considerable genetic heterogeneity. Next-generation sequencing of large cohorts has identified an increasing number of genes implicated in ID, but their roles in neurodevelopment remain largely unexplored. Here we report an ID syndrome caused by de novo heterozygous missense, nonsense, and frameshift mutations in BCL11A, encoding a transcription factor that is a putative member of the BAF swi/snf chromatin-remodeling complex. Using a comprehensive integrated approach to ID disease modeling, involving human cellular analyses coupled to mouse behavioral, neuroanatomical, and molecular phenotyping, we provide multiple lines of functional evidence for phenotypic effects. The etiological missense variants cluster in the amino-terminal region of human BCL11A, and we demonstrate that they all disrupt its localization, dimerization, and transcriptional regulatory activity, consistent with a loss of function. We show that Bcl11a haploinsufficiency in mice causes impaired cognition, abnormal social behavior, and microcephaly in accordance with the human phenotype. Furthermore, we identify shared aberrant transcriptional profiles in the cortex and hippocampus of these mouse models. Thus, our work implicates BCL11A haploinsufficiency in neurodevelopmental disorders and defines additional targets regulated by this gene, with broad relevance for our understanding of ID and related syndromes.

The Influence of Task Difficulty and Participant Age on Balance Control in ASD

Impairments in sensorimotor integration are reported in Autism Spectrum Disorder (ASD). Poor control of balance in challenging balance tasks is one suggested manifestation of these impairments, and is potentially related to ASD symptom severity. Reported balance and symptom severity relationships disregard age as a potential covariate, however, despite its involvement in balance development. We tested balance control during increasingly difficult balance conditions in children with ASD and typically developing peers, and investigated relationships between balance control and diagnostic/symptom severity metrics for participants with ASD, including age as a covariate. Balance deficits in ASD were exacerbated by stance alterations, but were not related to symptom severity when age was considered. These findings support impaired balance in ASD, especially in challenging conditions, but question a link between balance and symptom severity.