Automated syndrome diagnosis by three-dimensional facial imaging.

PURPOSE: Deep phenotyping is an emerging trend in precision medicine for genetic disease. The shape of the face is affected in 30-40% of known genetic syndromes. Here, we determine whether syndromes can be diagnosed from 3D images of human faces. METHODS: We analyzed variation in three-dimensional (3D) facial images of 7057 subjects: 3327 with 396 different syndromes, 727 of their relatives, and 3003 unrelated, unaffected subjects. We developed and tested machine learning and parametric approaches to automated syndrome diagnosis using 3D facial images. RESULTS: Unrelated, unaffected subjects were correctly classified with 96% accuracy. Considering both syndromic and unrelated, unaffected subjects together, balanced accuracy was 73% and mean sensitivity 49%. Excluding unrelated, unaffected subjects substantially improved both balanced accuracy (78.1%) and sensitivity (56.9%) of syndrome diagnosis. The best predictors of classification accuracy were phenotypic severity and facial distinctiveness of syndromes. Surprisingly, unaffected relatives of syndromic subjects were frequently classified as syndromic, often to the syndrome of their affected relative. CONCLUSION: Deep phenotyping by quantitative 3D facial imaging has considerable potential to facilitate syndrome diagnosis. Furthermore, 3D facial imaging of "unaffected" relatives may identify unrecognized cases or may reveal novel examples of semidominant inheritance.

Autoimmune vitiligo is associated with gain-of-function by a transcriptional regulator that elevates expression of HLA-A*02:01 in vivo.

HLA-A is a class I major histocompatibility complex receptor that presents peptide antigens on the surface of most cells. Vitiligo, an autoimmune disease in which skin melanocytes are destroyed by cognate T cells, is associated with variation in the HLA-A gene; specifically HLA-A*02:01, which presents multiple vitiligo melanocyte autoantigens. Refined genetic mapping localizes vitiligo risk in the HLA-A region to an SNP haplotype ∼20-kb downstream, spanning an ENCODE element with many characteristics of a transcriptional enhancer. Convergent CTCF insulator sites flanking the HLA-A gene promoter and the predicted transcriptional regulator, with apparent interaction between these sites, suggests this element regulates the HLA-A promoter. Peripheral blood mononuclear cells from healthy subjects homozygous for the high-risk haplotype expressed 39% more HLA-A RNA than cells from subjects carrying nonhigh-risk haplotypes (P = 0.0048). Similarly, RNAseq analysis of 1,000 Genomes Project data showed more HLA-A mRNA expressed in subjects homozygous for the high-risk allele of lead SNP rs60131261 than subjects homozygous for the low-risk allele (P = 0.006). Reporter plasmid transfection and genomic run-on sequence analyses confirm that the HLA-A transcriptional regulator contains multiple bidirectional promoters, with greatest activity on the high-risk haplotype, although it does not behave as a classic enhancer. Vitiligo risk associated with the MHC class I region thus derives from combined quantitative and qualitative phenomena: a SNP haplotype in a transcriptional regulator that induces gain-of-function, elevating expression of HLA-A RNA in vivo, in strong linkage disequilibrium with an HLA-A allele that confers *02:01 specificity.

Genomewide Association Study of African Children Identifies Association of SCHIP1 and PDE8A with Facial Size and Shape.

The human face is a complex assemblage of highly variable yet clearly heritable anatomic structures that together make each of us unique, distinguishable, and recognizable. Relatively little is known about the genetic underpinnings of normal human facial variation. To address this, we carried out a large genomewide association study and two independent replication studies of Bantu African children and adolescents from Mwanza, Tanzania, a region that is both genetically and environmentally relatively homogeneous. We tested for genetic association of facial shape and size phenotypes derived from 3D imaging and automated landmarking of standard facial morphometric points. SNPs within genes SCHIP1 and PDE8A were associated with measures of facial size in both the GWAS and replication cohorts and passed a stringent genomewide significance threshold adjusted for multiple testing of 34 correlated traits. For both SCHIP1 and PDE8A, we demonstrated clear expression in the developing mouse face by both whole-mount in situ hybridization and RNA-seq, supporting their involvement in facial morphogenesis. Ten additional loci demonstrated suggestive association with various measures of facial shape. Our findings, which differ from those in previous studies of European-derived whites, augment understanding of the genetic basis of normal facial development, and provide insights relevant to both human disease and forensics.

Genome-Wide Association Study Reveals Multiple Loci Influencing Normal Human Facial Morphology.

Numerous lines of evidence point to a genetic basis for facial morphology in humans, yet little is known about how specific genetic variants relate to the phenotypic expression of many common facial features. We conducted genome-wide association meta-analyses of 20 quantitative facial measurements derived from the 3D surface images of 3118 healthy individuals of European ancestry belonging to two US cohorts. Analyses were performed on just under one million genotyped SNPs (Illumina OmniExpress+Exome v1.2 array) imputed to the 1000 Genomes reference panel (Phase 3). We observed genome-wide significant associations (p < 5 x 10-8) for cranial base width at 14q21.1 and 20q12, intercanthal width at 1p13.3 and Xq13.2, nasal width at 20p11.22, nasal ala length at 14q11.2, and upper facial depth at 11q22.1. Several genes in the associated regions are known to play roles in craniofacial development or in syndromes affecting the face: MAFB, PAX9, MIPOL1, ALX3, HDAC8, and PAX1. We also tested genotype-phenotype associations reported in two previous genome-wide studies and found evidence of replication for nasal ala length and SNPs in CACNA2D3 and PRDM16. These results provide further evidence that common variants in regions harboring genes of known craniofacial function contribute to normal variation in human facial features. Improved understanding of the genes associated with facial morphology in healthy individuals can provide insights into the pathways and mechanisms controlling normal and abnormal facial morphogenesis.

Body size and allometric variation in facial shape in children.

OBJECTIVES: Morphological integration, or the tendency for covariation, is commonly seen in complex traits such as the human face. The effects of growth on shape, or allometry, represent a ubiquitous but poorly understood axis of integration. We address the question of to what extent age and measures of size converge on a single pattern of allometry for human facial shape. METHODS: Our study is based on two large cross-sectional cohorts of children, one from Tanzania and the other from the United States (N = 7,173). We employ 3D facial imaging and geometric morphometrics to relate facial shape to age and anthropometric measures. RESULTS: The two populations differ significantly in facial shape, but the magnitude of this difference is small relative to the variation within each group. Allometric variation for facial shape is similar in both populations, representing a small but significant proportion of total variation in facial shape. Different measures of size are associated with overlapping but statistically distinct aspects of shape variation. Only half of the size-related variation in facial shape can be explained by the first principal component of four size measures and age while the remainder associates distinctly with individual measures. CONCLUSIONS: Allometric variation in the human face is complex and should not be regarded as a singular effect. This finding has important implications for how size is treated in studies of human facial shape and for the developmental basis for allometric variation more generally.

Recessive progressive symmetric erythrokeratoderma results from a homozygous loss-of-function mutation of KRT83 and is allelic with dominant monilethrix.

BACKGROUND: Progressive symmetric erythrokeratoderma (PSEK) is a rare skin disorder characterised by symmetrically distributed demarcated hyperkeratotic plaques, often with associated palmoplantar hyperkeratosis, with new plaques appearing over time. Most cases are inherited in an autosomal dominant manner, although a few cases exhibit apparent autosomal recessive inheritance. OBJECTIVE: To identify the gene underlying autosomal recessive PSEK in a large Pakistani kindred. METHODS: We first carried out autozygosity mapping using microsatellite markers in candidate regions of the genome. We then carried out exome sequencing of five family members, autozygosity mapping and mutation analysis using the exome data and verification by Sanger sequencing. RESULTS: Autozygosity mapping and exome sequencing identified a homozygous frameshift deletion (c.811delA; p.Ser271fs) in KRT83, which co-segregated with the PSEK phenotype in the family and which is expected to abolish keratin 83, a type II keratin of hair and skin. CONCLUSIONS: At least some cases of PSEK result from loss-of-function mutations in KRT83. Heterozygous missense substitutions in KRT83 have been implicated in autosomal dominant monilethrix, a rare hair disorder. Our findings indicate that at least some cases of autosomal recessive PSEK and autosomal dominant monilethrix are allelic, respectively resulting from loss-of-function and missense mutations in the KRT83 gene. Together, these findings indicate that different types of mutations in KRT83 can result in quite different skin and hair phenotypes.

Rapid automated landmarking for morphometric analysis of three-dimensional facial scans.

Automated phenotyping is essential for the creation of large, highly standardized datasets from anatomical imaging data. Such datasets can support large-scale studies of complex traits or clinical studies related to precision medicine or clinical trials. We have developed a method that generates three-dimensional landmark data that meet the requirements of standard geometric morphometric analyses. The method is robust and can be implemented without high-performance computing resources. We validated the method using both direct comparison to manual landmarking on the same individuals and also analyses of the variation patterns and outlier patterns in a large dataset of automated and manual landmark data. Direct comparison of manual and automated landmarks reveals that automated landmark data are less variable, but more highly integrated and reproducible. Automated data produce covariation structure that closely resembles that of manual landmarks. We further find that while our method does produce some landmarking errors, they tend to be readily detectable and can be fixed by adjusting parameters used in the registration and control-point steps. Data generated using the method described here have been successfully used to study the genomic architecture of facial shape in two different genome-wide association studies of facial shape.

Multiplex SNaPshot-a new simple and efficient CYP2D6 and ADRB1 genotyping method.

BACKGROUND: Reliable, inexpensive, high-throughput genotyping methods are required for clinical trials. Traditional assays require numerous enzyme digestions or are too expensive for large sample volumes. Our objective was to develop an inexpensive, efficient, and reliable assay for CYP2D6 and ADRB1 accounting for numerous polymorphisms including gene duplications. MATERIALS AND METHODS: We utilized the multiplex SNaPshot® custom genotype method to genotype CYP2D6 and ADRB1. We compared the method to reference standards genotyped using the Taqman Copy Number Variant Assay followed by pyrosequencing quantification and determined assigned genotype concordance. RESULTS: We genotyped 119 subjects. Seven (5.9 %) were found to be CYP2D6 poor metabolizers (PMs), 18 (15.1 %) intermediate metabolizers (IMs), 89 (74.8 %) extensive metabolizers (EMs), and 5 (4.2 %) ultra-rapid metabolizers (UMs). We genotyped two variants in the ß1-adrenoreceptor, rs1801253 (Gly389Arg) and rs1801252 (Ser49Gly). The Gly389Arg genotype is Gly/Gly 18 (15.1 %), Gly/Arg 58 (48.7 %), and Arg/Arg 43 (36.1 %). The Ser49Gly genotype is Ser/Ser 82 (68.9 %), Ser/Gly 32 (26.9), and Gly/Gly 5 (4.2 %). The multiplex SNaPshot method was concordant with genotypes in reference samples. CONCLUSIONS: The multiplex SNaPshot method allows for specific and accurate detection of CYP2D6 genotypes and ADRB1 genotypes and haplotypes. This platform is simple and efficient and suited for high throughput.

NLRP1 haplotypes associated with vitiligo and autoimmunity increase interleukin-1ß processing via the NLRP1 inflammasome.

Nuclear localization leucine-rich-repeat protein 1 (NLRP1) is a key regulator of the innate immune system, particularly in the skin where, in response to molecular triggers such as pathogen-associated or damage-associated molecular patterns, the NLRP1 inflammasome promotes caspase-1-dependent processing of bioactive interleukin-1ß (IL-1ß), resulting in IL-1ß secretion and downstream inflammatory responses. NLRP1 is genetically associated with risk of several autoimmune diseases including generalized vitiligo, Addison disease, type 1 diabetes, rheumatoid arthritis, and others. Here we identify a repertoire of variation in NLRP1 by deep DNA resequencing. Predicted functional variations in NLRP1 reside in several common high-risk haplotypes that differ from the reference by multiple nonsynonymous substitutions. The haplotypes that are high risk for disease share two substitutions, L155H and M1184V, and are inherited largely intact due to extensive linkage disequilibrium across the region. Functionally, we found that peripheral blood monocytes from healthy subjects homozygous for the predominant high-risk haplotype 2A processed significantly greater (P < 0.0001) amounts of the IL-1ß precursor to mature bioactive IL-1ß under basal (resting) conditions and in response to Toll-like receptor (TLR) agonists (TLR2 and TLR4) compared with monocytes from subjects homozygous for the reference haplotype 1. The increase in basal release was 1.8-fold greater in haplotype 2A monocytes, and these differences between the two haplotypes were consistently observed three times over a 3-mo period; no differences were observed for IL-1α or TNFα. NLRP1 RNA and protein levels were not altered by the predominant high-risk haplotype, indicating that altered function of the corresponding multivariant NLRP1 polypeptide predisposes to autoimmune diseases by activation of the NLRP1 inflammasome.

Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element.

BACKGROUND: Ribosomal protein L30 belongs to the L7Ae family of RNA-binding proteins, which recognize diverse targets. L30 binds to kink-turn motifs in the 28S ribosomal RNA, L30 pre-mRNA, and mature L30 mRNA. L30 has a noncanonical function as a component of the UGA recoding machinery that incorporates selenocysteine (Sec) into selenoproteins during translation. L30 binds to a putative kink-turn motif in the Sec Insertion Sequence (SECIS) element in the 3' UTR of mammalian selenoprotein mRNAs. The SECIS also interacts with SECIS-binding protein 2 (SBP2), an essential factor for Sec incorporation. Previous studies showed that L30 and SBP2 compete for binding to the SECIS in vitro. The SBP2:SECIS interaction has been characterized but much less is known about how L30 recognizes the SECIS. RESULTS: Here we use enzymatic RNA footprinting to define the L30 binding site on the SECIS. Like SBP2, L30 protects nucleotides in the 5' side of the internal loop, the 5' side of the lower helix, and the SECIS core, including the GA tandem base pairs that are predicted to form a kink-turn. However, L30 has additional determinants for binding as it also protects nucleotides in the 3' side of the internal loop, which are not protected by SBP2. In support of the competitive binding model, we found that purified L30 repressed UGA recoding in an in vitro translation system, and that this inhibition was rescued by SBP2. To define the amino acid requirements for SECIS-binding, site-specific mutations in L30 were generated based on published structural studies of this protein in a complex with its canonical target, the L30 pre-mRNA. We identified point mutations that selectively inhibited binding of L30 to the SECIS, to the L30 pre-mRNA, or both RNAs, suggesting that there are subtle differences in how L30 interacts with the two targets. CONCLUSIONS: This study establishes that L30 and SBP2 bind to overlapping but non-identical sites on the SECIS. The amino acid requirements for the interaction of L30 with the SECIS differ from those that mediate binding to the L30 pre-mRNA. Our results provide insight into how L7Ae family members recognize their cognate RNAs.

Systematic replication of smoking disease associations using survey responses and EHR data in the All of Us Research Program.

OBJECTIVE: The All of Us Research Program (All of Us) aims to recruit over a million participants to further precision medicine. Essential to the verification of biobanks is a replication of known associations to establish validity. Here, we evaluated how well All of Us data replicated known cigarette smoking associations. MATERIALS AND METHODS: We defined smoking exposure as follows: (1) an EHR Smoking exposure that used International Classification of Disease codes; (2) participant provided information (PPI) Ever Smoking; and, (3) PPI Current Smoking, both from the lifestyle survey. We performed a phenome-wide association study (PheWAS) for each smoking exposure measurement type. For each, we compared the effect sizes derived from the PheWAS to published meta-analyses that studied cigarette smoking from PubMed. We defined two levels of replication of meta-analyses: (1) nominally replicated: which required agreement of direction of effect size, and (2) fully replicated: which required overlap of confidence intervals. RESULTS: PheWASes with EHR Smoking, PPI Ever Smoking, and PPI Current Smoking revealed 736, 492, and 639 phenome-wide significant associations, respectively. We identified 165 meta-analyses representing 99 distinct phenotypes that could be matched to EHR phenotypes. At P < .05, 74 were nominally replicated and 55 were fully replicated. At P < 2.68 × 10-5 (Bonferroni threshold), 58 were nominally replicated and 40 were fully replicated. DISCUSSION: Most phenotypes found in published meta-analyses associated with smoking were nominally replicated in All of Us. Both survey and EHR definitions for smoking produced similar results. CONCLUSION: This study demonstrated the feasibility of studying common exposures using All of Us data.

Parallel multiplicative target screening against divergent bacterial replicases: identification of specific inhibitors with broad spectrum potential.

Typically, biochemical screens that employ pure macromolecular components focus on single targets or a small number of interacting components. Researches rely on whole cell screens for more complex systems. Bacterial DNA replicases contain multiple subunits that change interactions with each stage of a complex reaction. Thus, the actual number of targets is a multiple of the proteins involved. It is estimated that the overall replication reaction includes up to 100 essential targets, many suitable for discovery of antibacterial inhibitors. We have developed an assay, using purified protein components, in which inhibitors of any of the essential targets can be detected through a common readout. Use of purified components allows each protein to be set within the linear range where the readout is proportional to the extent of inhibition of the target. By performing assays against replicases from model Gram-negative and Gram-positive bacteria in parallel, we show that it is possible to distinguish compounds that inhibit only a single bacterial replicase from those that exhibit broad spectrum potential.

Early-onset autoimmune vitiligo associated with an enhancer variant haplotype that upregulates class II HLA expression.

Vitiligo is an autoimmune disease in which melanocyte destruction causes skin depigmentation, with 49 loci known from previous GWAS. Aiming to define vitiligo subtypes, we discovered that age-of-onset is bimodal; one-third of cases have early onset (mean 10.3 years) and two-thirds later onset (mean 34.0 years). In the early-onset subgroup we found novel association with MHC class II region indel rs145954018, and independent association with the principal MHC class II locus from previous GWAS, represented by rs9271597; greatest association was with rs145954018del-rs9271597A haplotype (P = 2.40 × 10-86, OR = 8.10). Both rs145954018 and rs9271597 are located within lymphoid-specific enhancers, and the rs145954018del-rs9271597A haplotype is specifically associated with increased expression of HLA-DQB1 mRNA and HLA-DQ protein by monocytes and dendritic cells. Thus, for vitiligo, MHC regulatory variation confers extreme risk, more important than HLA coding variation. MHC regulatory variation may represent a significant component of genetic risk for other autoimmune diseases.

Human Facial Shape and Size Heritability and Genetic Correlations.

The human face is an array of variable physical features that together make each of us unique and distinguishable. Striking familial facial similarities underscore a genetic component, but little is known of the genes that underlie facial shape differences. Numerous studies have estimated facial shape heritability using various methods. Here, we used advanced three-dimensional imaging technology and quantitative human genetics analysis to estimate narrow-sense heritability, heritability explained by common genetic variation, and pairwise genetic correlations of 38 measures of facial shape and size in normal African Bantu children from Tanzania. Specifically, we fit a linear mixed model of genetic relatedness between close and distant relatives to jointly estimate variance components that correspond to heritability explained by genome-wide common genetic variation and variance explained by uncaptured genetic variation, the sum representing total narrow-sense heritability. Our significant estimates for narrow-sense heritability of specific facial traits range from 28 to 67%, with horizontal measures being slightly more heritable than vertical or depth measures. Furthermore, for over half of facial traits, >90% of narrow-sense heritability can be explained by common genetic variation. We also find high absolute genetic correlation between most traits, indicating large overlap in underlying genetic loci. Not surprisingly, traits measured in the same physical orientation (i.e., both horizontal or both vertical) have high positive genetic correlations, whereas traits in opposite orientations have high negative correlations. The complex genetic architecture of facial shape informs our understanding of the intricate relationships among different facial features as well as overall facial development.

Genome-wide association studies of autoimmune vitiligo identify 23 new risk loci and highlight key pathways and regulatory variants.

Vitiligo is an autoimmune disease in which depigmented skin results from the destruction of melanocytes, with epidemiological association with other autoimmune diseases. In previous linkage and genome-wide association studies (GWAS1 and GWAS2), we identified 27 vitiligo susceptibility loci in patients of European ancestry. We carried out a third GWAS (GWAS3) in European-ancestry subjects, with augmented GWAS1 and GWAS2 controls, genome-wide imputation, and meta-analysis of all three GWAS, followed by an independent replication. The combined analyses, with 4,680 cases and 39,586 controls, identified 23 new significantly associated loci and 7 suggestive loci. Most encode immune and apoptotic regulators, with some also associated with other autoimmune diseases, as well as several melanocyte regulators. Bioinformatic analyses indicate a predominance of causal regulatory variation, some of which corresponds to expression quantitative trait loci (eQTLs) at these loci. Together, the identified genes provide a framework for the genetic architecture and pathobiology of vitiligo, highlight relationships with other autoimmune diseases and melanoma, and offer potential targets for treatment.

Genome-wide association analyses identify 13 new susceptibility loci for generalized vitiligo.

We previously reported a genome-wide association study (GWAS) identifying 14 susceptibility loci for generalized vitiligo. We report here a second GWAS (450 individuals with vitiligo (cases) and 3,182 controls), an independent replication study (1,440 cases and 1,316 controls) and a meta-analysis (3,187 cases and 6,723 controls) identifying 13 additional vitiligo-associated loci. These include OCA2-HERC2 (combined P = 3.80 × 10(-8)), MC1R (P = 1.82 × 10(-13)), a region near TYR (P = 1.57 × 10(-13)), IFIH1 (P = 4.91 × 10(-15)), CD80 (P = 3.78 × 10(-10)), CLNK (P = 1.56 × 10(-8)), BACH2 (P = 2.53 × 10(-8)), SLA (P = 1.58 × 10(-8)), CASP7 (P = 3.56 × 10(-8)), CD44 (P = 1.78 × 10(-9)), IKZF4 (P = 2.75 × 10(-14)), SH2B3 (P = 3.54 × 10(-18)) and TOB2 (P = 6.81 × 10(-10)). Most vitiligo susceptibility loci encode immunoregulatory proteins or melanocyte components that likely mediate immune targeting and the relationships among vitiligo, melanoma, and eye, skin and hair coloration.

A novel protein phosphatase is a binding partner for the protein kinase domains of UNC-89 (Obscurin) in Caenorhabditis elegans.

Mutation of the Caenorhabditis elegans gene unc-89 results in disorganization of muscle A-bands. unc-89 encodes a giant polypeptide (900 kDa) containing two protein kinase domains, PK1 and PK2. Yeast two-hybrid screening using a portion of UNC-89 including PK2, yielded SCPL-1 (small CTD phosphatase-like-1), which contains a C terminal domain (CTD) phosphatase type domain. In addition to the PK2 domain, interaction with SCPL-1 required the putative autoinhibitory sequence, and immunoglobulin (Ig) and fibronectin type 3 (Fn3) domains lying N-terminal of the kinase domain. SCPL-1 also interacts with PK1, and it similarly requires the kinase domain and upstream Fn3 and Ig domains. Analogous regions from the two other giant kinases of C. elegans, twitchin and TTN-1, failed to interact with SCPL-1. The interaction between SCPL-1 and either Ig-Fn3-PK2 or Fn3-Ig-PK1 was confirmed by biochemical methods. The scpl-1b promoter is expressed in the same set of muscles as unc-89. Antibodies to SCPL-1 localize to the M-line and a portion of the I-band. Bacterially expressed SCPL-1 proteins have phosphatase activity in vitro with properties similar to previously characterized members of the CTD phosphatase family. RNA interference knockdown results in a defect in the function of egg-laying muscles. These studies suggest a new role for the CTD phosphatase family, that is, in muscle giant kinase signaling.