Phenotype and association results for facial factor 9.

<p>(A) Face showing the linear distances (in dark yellow) associated with factor 9; (B) LocusZoom plot showing the association (left y-axis; log10-transformed p-values) with factor 9. Genotyped SNPs are depicted by stars and imputed SNPs are depicted by circles. Shading of the points represents the linkage disequilibrium (r², based on the 1000 Genomes Project Europeans; gray indicates unknown LD) between each SNP and the top SNP, indicated by purple shading. The blue overlay shows the recombination rate (right y-axis). Positions of genes are shown below the plot. Note, gray points near the lead SNP are insertion-deletion variants in high LD (r2 = 0.91 and 0.77) with the lead SNP in our cohort.</p

Phenotype and association results for facial factor 14.

<p>(A) Face showing the linear distances (in red) associated with factor 14; (B) LocusZoom plot showing the association (left y-axis; log10-transformed p-values) with factor 14. Genotyped SNPs are depicted by stars and imputed SNPs are depicted by circles. Shading of the points represents the linkage disequilibrium (r², based on the 1000 Genomes Project Europeans; gray indicates unknown LD) between each SNP and the top SNP, indicated by purple shading. The blue overlay shows the recombination rate (right y-axis). Positions of genes are shown below the plot. Note, the gray point near the lead SNP is an insertion-deletion variant in high LD (r2 = 0.97) with the lead SNP in our cohort.</p

Genome-wide association study of facial morphology reveals novel associations with <i>FREM1</i> and <i>PARK2</i>

<div><p>Several studies have now shown evidence of association between common genetic variants and quantitative facial traits in humans. The reported associations generally involve simple univariate measures and likely represent only a small fraction of the genetic loci influencing facial morphology. In this study, we applied factor analysis to a set of 276 facial linear distances derived from 3D facial surface images of 2187 unrelated individuals of European ancestry. We retained 23 facial factors, which we then tested for genetic associations using a genome-wide panel of 10,677,593 single nucleotide polymorphisms (SNPs). In total, we identified genome-wide significant (p < 5 × 10⁻⁸) associations in three regions, including two that are novel: one involving measures of midface height at 6q26 within an intron of <i>PARK2</i> (lead SNP rs9456748; p = 4.99 × 10⁻⁸) and another involving measures of central upper lip height at 9p22 within <i>FREM1</i> (lead SNP rs72713618; p = 2.02 × 10⁻⁸). In both cases, the genetic association was stronger with the composite facial factor phenotype than with any of the individual linear distances that comprise those factors. While the biological role of <i>PARK2</i> in the craniofacial complex is currently unclear, there is evidence from both mouse models and Mendelian syndromes that <i>FREM1</i> may influence facial variation. These results highlight the potential value of data-driven multivariate phenotyping for genetic studies of human facial morphology.</p></div

Description of the 23 extracted factors describing facial morphology.

<p>Description of the 23 extracted factors describing facial morphology.</p

Structural enamel defects in third molars from a patient with PC-causing mutation in <i>KRT6A</i>.

<p>(<b>A</b>) Chromatograph on the left shows wild type sequence of <i>KRT6A</i> exon 1 at position c.505–519. Chromatograph on the right shows equivalent region for a PC patient with heterozygous mutation c.513C>A leading to missense mutation p.Asn171Lys (K6a^N171K). Schematics in lower panel shows the position of the N171K amino acid substitution at the beginning of the rod domain in K6a. (<b>B</b>) Micro-computed tomography analysis of a wisdom tooth extracted from a PC patient who is heterozygous for the mutation described in A. The upper panel shows the 3D reconstruction of the tooth crown. The bottom panel shows 3-axes sections of the molar on which enamel (E) and dentin (D) can be distinguished. (<b>C</b>) Scanning electron microscopy analysis of polished transverse section of wisdom teeth from PC and control patients. Sections were taken in the area of the cusps and in a plane perpendicular to the orientation of the enamel rods. Scale bar: 10 μm. (<b>D</b>) Differential interference contrast imaging of insoluble organic material isolated from a third molar from PC and control patients. White arrowheads indicate curls formed by the rod sheaths. Scale bar: 50 μm. (<b>E</b>) Immunohistochemical detection of K6 (red) on polished section of human third molar extracted from PC and control patients. White arrowheads indicate aggregates. Scale bar: 50 μm. DEJ, dentin-enamel junction.</p

Genetic association between missense SNPs in <i>KRT6B</i> and tooth decay experience.

<p>Genotype frequencies and quantification of caries experience per genotype for missense SNPs rs144860693 (<b>A</b>), rs28538343 (<b>B</b>) and rs61746354 (<b>C</b>). Pie charts on the left show the frequencies of all three genotypes for each SNP in the cohorts of 573 adults and 449 children that were evaluated for their caries experience. Bar graphs on the right represent the average and SEM (error bar) measures of three indices standardly used to assess caries experience: left, the number of tooth surfaces with untreated decay (DS and ds); center, the number of decayed, missing due to decay, and filled surfaces (DMFS and dfs); right, partial DMFS and dfs indices limited to molars and premolars pit and fissure surfaces (PF_DMFS and pf_dfs).</p

K6 proteins in enamel and genetic association between missense SNPs in <i>KRT6</i> genes and tooth decay experience.

<p>(<b>A</b>) RNA-seq data from mouse enamel organ showing expression of <i>Krt6a</i>, <i>Krt6b</i>, <i>Krt16</i> and <i>Krt17</i> in the tissue. The schematics at the top shows a mouse mandible at postnatal day 10 from which the enamel organ (red) was micro-dissected for RNA extraction. Tracks represent the alignment of the RNA-seq reads to the mouse genome for the <i>Krt6a</i>, <i>Krt6b</i>, <i>Krt16</i> and <i>Krt17</i> loci. (<b>B</b>) Immunohistochemical analysis of K6 distribution in rat enamel organ at postnatal day 10. Anti-K6 antibody recognizes all members of the K6 family (K6a and K6b in rodents). Nuclei are stained with DAPI (blue). Inset at the top shows enlarged view of Tomes’ processes. PL, papillary layer; SI, stratum intermedium; Am, ameloblasts; TP, Tomes’ processes; R, rod; IR, interrod. Scale bar: 50 μm. (<b>C</b>) Immunohistochemical detection of K6 on polished sections of human third molars. Antibody raised in guinea pig against the N-terminal of K6 (recognizes K6a, K6b and K6c in humans) was used (green). As indicated by the schematics on the bottom right corner, the image in the left panel was acquired from mesial-distal sections of human third molars and shows the area of enamel adjacent to dentin, whereas the image in the right panel was acquired from transverse section of the crown near the cusps. DEJ, dentin-enamel junction. Scale bars: 100 μm for left panel, 20 μm for right panel. (<b>D</b>) Schematics showing the structure of keratin proteins with a central rod domain flanked by a head domain and a tail domain. Shown in red are the position of the five missense SNPs in <i>KRT6A</i>, <i>KRT6B</i> and <i>KRT6C</i> that were found to have significant genetic association with tooth decay risk as measured by three indices of dental caries experience. All SNPs associated with high caries experience in adults resulted in substitutions in the head domain of K6 proteins, while the unique SNP that showed association with tooth decay risk in children only results in a substitution in the tail domain of K6b (Y497C). Areas where mutations leading to PC have so far been identified are highlighted by black arrowheads and are exclusively at the beginning and at the end of the rod domain. (<b>E</b>) Linkage disequilibrium data measured by R² between the five missense polymorphisms highlighted in D.</p

Effects of S143N and N171K mutations on the assembly of K6a in ameloblast-like cells.

<p>(<b>A</b>) Alignment of K6a, K6b and K6c protein sequences between amino acids 138 and 175 showing the position of serine 143 and asparagine 171 on both sides of the transition between the head and the rod domain (black box labeled HR). Serine 143 is right upstream of an LL^<b>S</b><b>/</b>_<b>T</b>PL consensus phosphorylation site (blue circle labeled <b>P</b>) that is highly conserved in type II keratins. Serine 143 is also part of an <b>N</b>QSL potential N-glycosylation site (green circle labeled <b>G</b>). (<b>B</b>) Schematic representation of pBi4-GFP/K6a bidirectional constructs used for tetracycline inducible expression of GFP with <i>KRT6A</i>^<i>W</i>T, <i>KRT6A</i>^<i>G428A</i> (produces K6a^S143N) or <i>KRT6A</i>^<i>C513A</i> (produces K6a^N171K mutant). When transfected into cells expressing the transactivator rTA, transgene expression through binding of rTA to the tetracycline responsive element (TRE) can be induced by addition of doxycycline (Dox) to the culture medium. (<b>C</b>) Detection of GFP induction in ameloblasts-like cells stably expressing rTA (ALC-TetON) transfected with pBi4-GFP/K6a constructs and grown with or without Dox. (<b>D</b>) Immunohistochemical analysis of K6a subcellular distribution (red) in ALC-TetON cells producing K6a^WT, K6a^S143N or K6a^N171K. Nuclei are stained with DAPI (blue) and actin filaments are stained with phalloidin conjugated to Alexa Fluor 647 (white).</p

Genetic association of missense SNPs in <i>KRT6A</i>, <i>KRT6B</i> and <i>KRT6C</i> with dental caries in permanent dentition of adults (N = 573, 25–50 years) and primary dentition of children (N = 449, 6–12 years).

<p>Genetic association of missense SNPs in <i>KRT6A</i>, <i>KRT6B</i> and <i>KRT6C</i> with dental caries in permanent dentition of adults (N = 573, 25–50 years) and primary dentition of children (N = 449, 6–12 years).</p