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Variations in the form of the human face, which plays a role in our individual identities and societal interactions, have fascinated scientists and artists alike. Here, we review our current understanding of the genetics underlying variation in craniofacial morphology and disease-associated dysmorphology, synthesizing decades of progress on Mendelian syndromes in addition to more recent results from genome-wide association studies of human facial shape and disease risk. We also discuss the various approaches used to phenotype and quantify facial shape, which are of particular importance due to the complex, multipartite nature of the craniofacial form. We close by discussing how experimental studies have contributed and will further contribute to our understanding of human genetic variation and then proposing future directions and applications for the field. ispartof: ANNUAL REVIEW OF GENOMICS AND HUMAN GENETICS vol:23 pages:383-412 ispartof: location:United States status: published

Transcriptional regulation exhibits extensive robustness, but human genetics indicates sensitivity to transcription factor (TF) dosage. Reconciling such observations requires quantitative studies of TF dosage effects at trait-relevant ranges, largely lacking so far. TFs play central roles in both normal-range and disease-associated variation in craniofacial morphology; we therefore developed an approach to precisely modulate TF levels in human facial progenitor cells and applied it to SOX9, a TF associated with craniofacial variation and disease (Pierre Robin sequence (PRS)). Most SOX9-dependent regulatory elements (REs) are buffered against small decreases in SOX9 dosage, but REs directly and primarily regulated by SOX9 show heightened sensitivity to SOX9 dosage; these RE responses partially predict gene expression responses. Sensitive REs and genes preferentially affect functional chondrogenesis and PRS-like craniofacial shape variation. We propose that such REs and genes underlie the sensitivity of specific phenotypes to TF dosage, while buffering of other genes leads to robust, nonlinear dosage-to-phenotype relationships. ispartof: NATURE GENETICS vol:55 issue:5 ispartof: location:United States status: Published online

The cranial vault – the portion of the skull surrounding the brain and cerebellum – is highly variable, clinically relevant, and heritable, yet its genetic architecture remains poorly understood. Here, we conducted a joint multi-ancestry and admixed multivariate GWAS on 3D cranial vault shape extracted from magnetic resonance images of 6,772 children from the ABCD study cohort, identifying 30 genome-wide significant genetic loci and replicating 20 of these signals in 16,947 additional individuals of the UK Biobank. This joint multi-ancestry GWAS was enriched for genetic components of cranial vault shape shared across ancestral groups and yielded a greater discovery than a European-only GWAS. We present supporting evidence for parietal versus frontal bone localization for several of the identified genes based on expression patterns in E15.5 mice. Collectively, our GWAS loci were enriched for processes related to skeletal development and showed elevated activity in cranial neural crest cells, suggesting a role during early craniofacial development. Among the identified genes, wereRUNX2and several of its upstream and downstream actors, highlighting the prominent role of intramembranous ossification – which takes place at the cranial sutures – in influencing cranial vault shape. We found that mutations in many genes associated with craniosynostosis exert their pathogenicity by modulating the same pathways involved in normal cranial vault development. This was further demonstrated in a non-syndromic sagittal craniosynostosis case-parent trio dataset of 63 probands (n = 189), where our GWAS signals nearBMP2, BBS9, andZIC2contributed significantly to disease risk. Moreover, we found strong evidence of overlap with genes influencing the morphology of the face and the brain, suggesting a common genetic architecture connecting these developmentally adjacent structures. Overall, our study provides a comprehensive overview of the genetics underlying normal cranial vault shape and its relevance for understanding modern human craniofacial diversity and the etiology of congenital malformations.

Gene regulatory networks ensure that important genes are expressed at precise levels. When gene expression is sufficiently perturbed, it can lead to disease. To understand how gene expression disruptions percolate through a network, we must first map connections between regulatory genes and their downstream targets. However, we lack comprehensive knowledge of the upstream regulators of most genes. Here, we developed an approach for systematic discovery of upstream regulators of critical immune factors-IL2RA, IL-2 and CTLA4-in primary human T cells. Then, we mapped the network of the target genes of these regulators and putative cis-regulatory elements using CRISPR perturbations, RNA-seq and ATAC-seq. These regulators form densely interconnected networks with extensive feedback loops. Furthermore, this network is enriched for immune-associated disease variants and genes. These results provide insight into how immune-associated disease genes are regulated in T cells and broader principles about the structure of human gene regulatory networks.

Transcriptional regulation displays extensive robustness, but human genetics indicate sensitivity to transcription factor (TF) dosage. Reconciling such observations requires quantitative studies of TF dosage effects at trait-relevant ranges, which are lacking to date. TFs play central roles in both normal-range and disease-associated variation in facial morphology; we therefore developed an approach to precisely modulate TF levels in human facial progenitors and applied it to SOX9, a TF associated with craniofacial variation and disease (Pierre Robin Sequence, PRS). We found that most SOX9-dependent regulatory elements (REs) are buffered against small decreases in SOX9 dosage, but REs directly and primarily regulated by SOX9 show heightened sensitivity to SOX9 dosage; these RE responses predict gene expression responses. Sensitive REs and genes underlie the vulnerability of chondrogenesis and PRS-like craniofacial shape variation to SOX9 dosage perturbation. We propose that such REs and genes drive the sensitivity of specific phenotypes to TF dosage, while buffering of other genes leads to robust, nonlinear dosage-to-phenotype relationships.

Gene regulatory divergence is thought to play a central role in determining human-specific traits. However, our ability to link divergent regulation to divergent phenotypes is limited. Here, we utilized human-chimpanzee hybrid induced pluripotent stem cells to study gene expression separating these species. The tetraploid hybrid cells allowed us to separate cis- from trans-regulatory effects, and to control for non-genetic confounding factors. We differentiated these cells into cranial neural crest cells (CNCCs), the primary cell type giving rise to the face. We discovered evidence of lineage-specific selection on the hedgehog signaling pathway, including a human-specific 6-fold down-regulation of EVC2 (LIMBIN), a key hedgehog gene. Inducing a similar down-regulation of EVC2 substantially reduced hedgehog signaling output. Mice and humans lacking functional EVC2 show striking phenotypic parallels to human-chimpanzee craniofacial differences, suggesting that the regulatory divergence of hedgehog signaling may have contributed to the unique craniofacial morphology of humans.

The human face is complex and multipartite, and characterization of its genetic architecture remains challenging. Using a multivariate genome-wide association study meta-analysis of 8,246 European individuals, we identified 203 genome-wide-significant signals (120 also study-wide significant) associated with normal-range facial variation. Follow-up analyses indicate that the regions surrounding these signals are enriched for enhancer activity in cranial neural crest cells and craniofacial tissues, several regions harbor multiple signals with associations to different facial phenotypes, and there is evidence for potential coordinated actions of variants. In summary, our analyses provide insights into the understanding of how complex morphological traits are shaped by both individual and coordinated genetic actions. ispartof: Nature Genetics vol:53 issue:1 ispartof: location:United States status: Published online

Little is known about how human Y-Chromosome gene expression directly contributes to differences between XX (female) and XY (male) individuals in nonreproductive tissues. Here, we analyzed quantitative profiles of Y-Chromosome gene expression across 36 human tissues from hundreds of individuals. Although it is often said that Y-Chromosome genes are lowly expressed outside the testis, we report many instances of elevated Y-Chromosome gene expression in a nonreproductive tissue. A notable example is EIF1AY, which encodes eukaryotic translation initiation factor 1A Y-linked, together with its X-linked homolog EIF1AX. Evolutionary loss of a Y-linked microRNA target site enabled up-regulation of EIF1AY, but not of EIF1AX, in the heart. Consequently, this essential translation initiation factor is nearly twice as abundant in male as in female heart tissue at the protein level. Divergence between the X and Y Chromosomes in regulatory sequence can therefore lead to tissue-specific Y-Chromosome-driven sex biases in expression of critical, dosage-sensitive regulatory genes.

The skin color is one of the most diverse human traits and is determined by the quantity, type and distribution of melanin. Here, we leverage light scattering properties of melanin to conduct a genome-wide CRISPR-Cas9 screen for novel regulators of melanogenesis. We identify functionally diverse genes converging on melanosome biogenesis, endosomal transport and transcriptional/posttranscriptional gene regulation, most of which represent novel associations with pigmentation. A survey of transcriptomes from diversely pigmented individuals reveals that the majority of genes discovered in our screen are upregulated in dark skin melanocytes, in agreement with their melanin-promoting function and potential contribution to skin color variation. This association is further buttressed by the significant skin color heritability enrichment in the vicinity of these genes. Taken together, our study presents a novel approach to assay pigmentation and uncovers a plethora of melanogenesis regulators, with broad implications for human variation, cell biology and medicine.One Sentence SummaryGenetic screen uncovers genes involved in human melanogenesis, many of which are differentially expressed in individuals of diverse skin color.

Facial morphology is highly variable, both within and among human populations, and a sizable portion of this variation is attributable to genetics. Previous genome scans have revealed more than 100 genetic loci associated with different aspects of normal-range facial variation. Most of these loci have been detected in Europeans, with few studies focusing on other ancestral groups. Consequently, the degree to which facial traits share a common genetic basis across diverse sets of humans remains largely unknown. We therefore investigated the genetic basis of facial morphology in an East African cohort. We applied an open-ended data-driven phenotyping approach to a sample of 2,595 3D facial images collected on Tanzanian children. This approach segments the face into hierarchically arranged, multivariate features that capture the shape variation after adjusting for age, sex, height, weight, facial size and population stratification. Genome scans of these multivariate shape phenotypes revealed significant (p < 2.5 × 10−8) signals at 20 loci, which were enriched for active chromatin elements in human cranial neural crest cells and embryonic craniofacial tissue, consistent with an early developmental origin of the facial variation. Two of these associations were in highly conserved regions showing craniofacial-specific enhancer activity during embryological development (5q31.1 and 12q21.31). Six of the 20 loci surpassed a stricter threshold accounting for multiple phenotypes with study-wide significance (p < 6.25 × 10−10). Cross-population comparisons indicated 10 association signals were shared with Europeans (seven sharing the same associated SNP), and facilitated fine-mapping of causal variants at previously reported loci. Taken together, these results may point to both shared and population-specific components to the genetic architecture of facial variation., Author summary Genetic factors play an important role in shaping human facial features. Over the last decade, studies have identified numerous genes associated with various facial traits. The vast majority of these studies have focused on European or Asian populations, while African populations have been underrepresented. Increasing the diversity of these analyses can reveal novel associations and cross-population analyses can help deepen our understanding of known genetic associations. We therefore performed a genome scan of 3D facial features in African children from Tanzania and then compared our results to Europeans. We found 20 regions of the genome associated with facial shape in Tanzanian children, 10 of which were also present in Europeans, indicating evidence for a partly shared genetic basis for human facial shape across populations. In addition, about half of the genetic associations observed in Tanzanians were not present in Europeans, and some of the shared signals differed between populations in the specific genetic variants associated or specific facial traits affected. These results shed light on the shared and population-specific genetic contributors to normal-range facial variation.

SummaryComplex gene regulatory networks ensure that important genes are expressed at precise levels. When gene expression is sufficiently perturbed it can lead to disease. To understand how gene expression disruptions percolate through a network, we must first map connections between regulatory genes and their downstream targets. However, we lack comprehensive knowledge of the upstream regulators of most genes. Here we developed an approach for systematic discovery of upstream regulators of critical immune factors – IL2RA, IL-2, and CTLA4 – in primary human T cells. Then, we mapped the network of these regulators’ target genes and enhancers using CRISPR perturbations, RNA-Seq, and ATAC-Seq. These regulators form densely interconnected networks with extensive feedback loops. Furthermore, this network is highly enriched for immune-associated disease variants and genes. These results provide insight into how immune-associated disease genes are regulated in T cells and broader principles about the structure of human gene regulatory networks.HighlightsA systematic approach to identify upstream regulators of key immune genes in primary human cellsComprehensive RNA-Seq and ATAC-Seq perturbation maps after KO of individual discovered regulatorsAnalysis uncovers a highly interconnected regulatory network of enhancers and genes in T cellsThis network is highly enriched for immune disease variants and genes shedding light on the trans-regulatory connections among key immune genes in health and disease

Evidence from model organisms and clinical genetics suggests coordination between the developing brain and face, but the role of this link in common genetic variation remains unknown. We performed a multivariate genome-wide association study of cortical surface morphology in 19,644 individuals of European ancestry, identifying 472 genomic loci influencing brain shape, of which 76 are also linked to face shape. Shared loci include transcription factors involved in craniofacial development, as well as members of signaling pathways implicated in brain-face cross-talk. Brain shape heritability is equivalently enriched near regulatory regions active in either forebrain organoids or facial progenitors. However, we do not detect significant overlap between shared brain-face genome-wide association study signals and variants affecting behavioral-cognitive traits. These results suggest that early in embryogenesis, the face and brain mutually shape each other through both structural effects and paracrine signaling, but this interplay may not impact later brain development associated with cognitive function. A multivariate genome-wide association study highlighting loci that influence both face and brain shape suggesting shared developmental axes during early embryogenesis. These loci did not overlap with those governing behavioral-cognitive traits or neuropsychiatric risk indicating divergence between early brain development and cognitive function. J.W. was supported by the Howard Hughes Medical Institute, a Lorry Lokey endowed professorship and a Stinehart Reed award. S.N. was supported by a Helen Hay Whitney Fellowship. The KU Leuven research team and analyses were supported by the National Institutes of Health (NIH; 1-R01-DE027023 and 2-R01-DE027023), The Research Fund KU Leuven (BOF-C1, C14/15/081 and C14/20/081) and The Research Program of the Research Foundation in Flanders (FWO; G078518N). The computational resources and services used in this work were provided by the VSC (Flemish Supercomputer Center), funded by the FWO and the Flemish Government (department EWI). J.P.S. was supported by an NIH training grant (5T32HG000044-23). J.K.P. was supported by the NIH (HG008140 and HG009431). Pittsburgh personnel, data collection and analyses were supported by the National Institute of Dental and Craniofacial Research (U01-DE020078, R01-DE016148 and R01-DE027023). Funding for genotyping by the National Human Genome Research Institute (X01-HG007821 and X01-HG007485) and funding for initial genomic data cleaning by the University of Washington were provided by contract HHSN268201200008I from the National Institute for Dental and Craniofacial Research awarded to the Center for Inherited Disease Research (https://www.cidr.jhmi.edu/).J.T.was supported by the NIH (5R01-DA033431-07) and the National Science Foundation (1922598). This research has been conducted, in part, using the UKB resource under application no. 43193 (understanding the genetic architecture of human brain shape from MRI using global-to-local shape segmentations), and we are grateful for all the participants in that resource. This manuscript reflects the views of the authors and may not reflect the opinions or views of the UKB funders and investigators. Data used in the preparation of this article were obtained from the ABCD Study (https://abcdstudy.org), held in the National Institute of Mental Health Data Archive. This is a multisite, longitudinal study designed to recruit more than 10,000 children aged 9-10 years and follow them over 10 years into early adulthood. The ABCD Study is supported by the NIH and additional federal partners under award nos. U01DA041022, U01DA041028, U01DA041048, U01DA041089, U01DA041106, U01DA041117, U01DA041120, U01DA041134, U01DA041148, U01DA041156, U01DA041174, U24DA041123, U24DA041147, U01DA041093 and U01DA041025. A full list of supporters is available at https://abcdstudy.org/federal-partners.html.A list of participating sites and study investigators can be found at https://abcdstudy.org/wp-content/uploads/2019/04/Consortium_Members.pdf.ABCD consortium investigators provided data but did not necessarily participate in analysis or writing of this report. This manuscript reflects the views of the authors and may not reflect the opinions or views of the NIH or ABCD consortium investigators. Naqvi, S; Wysocka, J (corresponding author), Stanford Univ, Sch Med, Dept Chem & Syst Biol, Stanford, CA 94305 USA ; Stanford Univ, Sch Med, Dept Genet, Stanford, CA 94305 USA ; Stanford Univ, Sch Med, Dept Biol, Stanford, CA 94305 USA. Claes, P (corresponding author), Katholieke Univ Leuven, Dept Human Genet, Leuven, Belgium. ; Univ Hosp Leuven, Med Imaging Res Ctr, Leuven, Belgium ; Katholieke Univ Leuven, ESAT PSI, Dept Elect Engn, Leuven, Belgium ; Murdoch Childrens Res Inst, Melbourne, Vic, Australia. Wysocka, J (corresponding author), Stanford Univ, Sch Med, Dept Dev Biol, Stanford, CA 94305 USA ; Stanford Univ, Sch Med, Howard Hughes Med Inst, Stanford, CA 94305 USA.

Evidence from both model organisms and clinical genetics suggests close coordination between the developing brain and face1–8, but it remains unknown whether this developmental link extends to genetic variation that drives normal-range diversity of face and brain shape. Here, we performed a multivariate genome-wide association study of cortical surface morphology in 19,644 European-ancestry individuals and identified 472 genomic loci influencing brain shape at multiple levels. We discovered a substantial overlap of these brain shape association signals with those linked to facial shape variation, with 76 common to both. These shared loci include transcription factors with cell-intrinsic roles in craniofacial development, as well as members of signaling pathways involved in brain-face crosstalk. Brain shape heritability is equivalently enriched near regulatory regions active in either brain organoids or in facial progenitor cells. However, brain shape association signals shared with face shape are distinct from those shared with behavioral-cognitive traits or neuropsychiatric disorder risk. Together, we uncover common genetic variants and candidate molecular players underlying brain-face interactions. We propose that early in embryogenesis, the face and the brain mutually shape each other through a combination of structural effects and paracrine signaling, but this interplay may have little impact on later brain development associated with cognitive function.

The human face is complex and multipartite, and characterization of its genetic architecture remains intriguingly challenging. Applying GWAS to multivariate shape phenotypes, we identified 203 genomic regions associated with normal-range facial variation, 117 of which are novel. The associated regions are enriched for both genes relevant to craniofacial and limb morphogenesis and enhancer activity in cranial neural crest cells and craniofacial tissues. Genetic variants grouped by their contribution to similar aspects of facial variation show high within-group correlation of enhancer activity, and four SNP pairs display evidence of epistasis, indicating potentially coordinated actions of variants within the same cell types or tissues. In sum, our analyses provide new insights for understanding how complex morphological traits are shaped by both individual and coordinated genetic actions.

Mammalian primordial germ cells (PGCs) are induced in the embryonic epiblast, before migrating to the nascent gonads. In fish, frogs, and birds, the germline segregates even earlier, through the action of maternally inherited germ plasm. Across vertebrates, migrating PGCs retain a broad developmental potential, regardless of whether they were induced or maternally segregated. In mammals, this potential is indicated by expression of pluripotency factors, and the ability to generate teratomas and pluripotent cell lines. How the germline loses this developmental potential remains unknown. Our genome-wide analyses of embryonic human and mouse germlines reveal a conserved transcriptional program, initiated in PGCs after gonadal colonization, that differentiates germ cells from their germline precursors and from somatic lineages. Through genetic studies in mice and pigs, we demonstrate that one such gonad-induced factor, the RNA-binding protein DAZL, is necessary in vivo to restrict the developmental potential of the germline; DAZL’s absence prolongs expression of a Nanog pluripotency reporter, facilitates derivation of pluripotent cell lines, and causes spontaneous gonadal teratomas. Based on these observations in humans, mice, and pigs, we propose that germ cells are determined after gonadal colonization in mammals. We suggest that germ cell determination was induced late in embryogenesis—after organogenesis has begun—in the common ancestor of all vertebrates, as in modern mammals, where this transition is induced by somatic cells of the gonad. We suggest that failure of this process of germ cell determination likely accounts for the origin of human testis cancer.

The analysis of contemporary genomic data typically operates on one-dimensional phenotypic measurements (e.g. standing height). Here we report on a data-driven, family-informed strategy to facial phenotyping that searches for biologically relevant traits and reduces multivariate 3D facial shape variability into amendable univariate measurements, while preserving its structurally complex nature. We performed a biometric identification of siblings in a sample of 424 children, defining 1,048 sib-shared facial traits. Subsequent quantification and analyses in an independent European cohort (n = 8,246) demonstrated significant heritability for a subset of traits (0.17–0.53) and highlighted 218 genome-wide significant loci (38 also study-wide) associated with facial variation shared by siblings. These loci showed preferential enrichment for active chromatin marks in cranial neural crest cells and embryonic craniofacial tissues and several regions harbor putative craniofacial genes, thereby enhancing our knowledge on the genetic architecture of normal-range facial variation., Author summary The human face is a highly variable trait composed of distinct features, each influenced by genetic and environmental factors. The strong genetic component is primarily evidenced by the facial similarity between identical twins and the clear facial resemblances within families. Over the past decade, a powerful methodological toolbox of computational and statistical genetics has been developed to study the genetic architecture of complex traits. However, these methods typically require one-dimensional phenotypic measurements (e.g. width of the nose or spacing between the eyes) that fail to accurately describe 3D facial shape. In this paper, we learn from 3D facial data itself, a series of relevant traits that are guided by the facial similarity observed between sibling pairs. Importantly, while preserving the structural convolution of the face, these traits also fit the requirements as input to the well-established statistical tools. In doing so, we have identified many genetic loci that are associated with a wide range of facial features. Some of these regions contained genes related to embryonic facial development, and craniofacial malformations. An improved understanding of the genetic basis of facial shape can have several important applications, for example in developmental biology, medical genetics and forensic sciences.

Sex differences abound in human health and disease, as they do in other mammals used as models. The extent to which sex differences are conserved at the molecular level across species and tissues is unknown. We surveyed sex differences in gene expression in human, macaque, mouse, rat, and dog, across 12 tissues. In each tissue, we identified hundreds of genes with conserved sex-biased expression-findings that, combined with genomic analyses of human height, explain ∼12% of the difference in height between females and males. We surmise that conserved sex biases in expression of genes otherwise operating equivalently in females and males contribute to sex differences in traits. However, most sex-biased expression arose during the mammalian radiation, which suggests that careful attention to interspecies divergence is needed when modeling human sex differences., National Institutes of Health (U.S.) (Grant R01HG007852), National Institutes of Health (U.S.) (Grant U01HG007857)

Mammalian X and Y chromosomes evolved from an ordinary autosomal pair. Genetic decay of the Y led to X chromosome inactivation (XCI) in females, but some Y-linked genes were retained during the course of sex chromosome evolution, and many X-linked genes did not become subject to XCI. We reconstructed gene-by-gene dosage sensitivities on the ancestral autosomes through phylogenetic analysis of microRNA (miRNA) target sites and compared these preexisting characteristics to the current status of Y-linked and X-linked genes in mammals. Preexisting heterogeneities in dosage sensitivity, manifesting as differences in the extent of miRNA-mediated repression, predicted either the retention of a Y homolog or the acquisition of XCI following Y gene decay. Analogous heterogeneities among avian Z-linked genes predicted either the retention of a W homolog or gene-specific dosage compensation following W gene decay. Genome-wide analyses of human copy number variation indicate that these heterogeneities consisted of sensitivity to both increases and decreases in dosage. We propose a model of XY/ZW evolution incorporating such preexisting dosage sensitivities in determining the evolutionary fates of individual genes. Our findings thus provide a more complete view of the role of dosage sensitivity in shaping the mammalian and avian sex chromosomes, and reveal an important role for post-transcriptional regulatory sequences (miRNA target sites) in sex chromosome evolution.

Cooperativity arising from local interactions in equilibrium receptor systems provides gain, but does not increase sensory performance, as measured by the signal-to-noise ratio (SNR) due to a fundamental tradeoff between gain and intrinsic noise. Here we allow sensing to be a nonequilibrium process and show that energy dissipation cannot circumvent the fundamental tradeoff, so that the SNR is still optimal for independent receptors. For systems requiring high gain, nonequilibrium 2D-coupled receptors maximize the SNR, revealing a new design principle for biological sensors.