Transcriptome Analysis of Gingival Tissues of Enamel&#X2010

Received: 7 January 2019 | Revised: 8 April 2019 | Accepted: 12 April 2019 DOI: 10.1111/jre.12666

ORIGINAL ARTICLE

Transcriptome analysis of gingival tissues of enamel‐renal syndrome

Yi‐Ping Wang1,2 | Hung‐Ying Lin3 | Wen‐Lan Zhong1 | James P. Simmer4 | Shih‐Kai Wang1,5

1Department of Dentistry, School of Dentistry, National Taiwan University, Taipei Background and objective: Biallelic loss‐of‐function mutations of human City, Taiwan FAM20A have been known to cause enamel‐renal syndrome (ERS), featured by 2 Graduate Institute of Clinical Dentistry, agenesis of dental enamel, nephrocalcinosis, and other orodental abnormalities, School of Dentistry, National Taiwan University, Taipei City, Taiwan including gingival hyperplasia. However, while the histopathology of this gingival 3Department of Oral and Maxillofacial anomaly has been analyzed, its underlying molecular mechanism remains largely Surgery, National Taiwan University unknown. This study aimed to unravel the pathogenesis of gingival hyperplasia Hospital, Taipei City, Taiwan 4Department of Biologic and Materials in ERS. Sciences, University of Michigan School of Methods: Whole‐exome sequencing was conducted for an ERS case. Transcriptome Dentistry, Ann Arbor, Michigan analyses, using RNA sequencing, of the patient's gingiva were performed to unravel 5Department of Pediatric Dentistry, National Taiwan University Children’s Hospital, Taipei dysregulated molecules and aberrant biological processes underlying the gingival pa‐ City, Taiwan thology of ERS, which was further confirmed by histology and immunohistochemistry.

Correspondence Results: Two novel frameshift FAM20A mutations in Exon 1 (g.5417delG; c.129delG; Shih‐Kai Wang, Department of Dentistry, p.Cys44Alafs*101) and Exon 5 (g.62248_62249delAG; c.734_735delAG; National Taiwan University School of Dentistry, No.1, Changde St., Taipei City p.Glu245Glyfs*11) were identified. Transcriptional profiling of patient's gingival tissue 10048, Taiwan. revealed a total of 1683 genes whose expression had increased (1129 genes) or de‐ Email: [email protected] creased (554 genes) at least 2‐fold compared to control gingival tissues. There were Funding information 951 gene ontology (GO) terms of biological process being significantly over‐repre‐ National Institute of Dental and Craniofacial Research, Grant/Award Number: DE015846 sented or under‐represented. While GOs involved in extracellular matrix organization, and DE027675; Ministry of Science and angiogenesis, biomineralization, and epithelial cell proliferation appeared to be acti‐ Technology, Taiwan, Grant/Award Number: Grant 107-2314-B-002-014; National vated in ERS gingiva, genes related to keratinocyte differentiation, epithelial develop‐ Taiwan University Hospital, Grant/Award ment, and keratinization were of decreased expression. FAM20A immunohistochemistry Number: Grant 106-N3424 revealed a strong reactivity at the suprabasal layers of epithelium in control gingiva but showed a significantly diminished and scattered signal in ERS tissues. For genes show‐ ing significant over‐expression in the transcriptome analyses, namely ALPL, SPARC, and ACTA2, an increased immunoreactivity was observed. Conclusion: Our results unraveled a potential role for FAM20A in homeostasis of both gingival epithelium and connective tissues.

KEYWORDS enamel, gingival hyperplasia, RNA sequencing, transcriptome

1 | INTRODUCTION 2 | MATERIAL AND METHODS

Amelogenesis imperfecta type IG (AI1G; OMIM #204690), better 2.1 | Subject recruitment and mutational analyses known as enamel‐renal syndrome (ERS), is an autosomal recessive The human study protocol and subject consents were reviewed and disorder characterized by severe enamel hypoplasia/aplasia (thin or approved by the Institutional Review Boards at the National Taiwan no dental enamel), delayed or failed tooth eruption, misshapen teeth University Hospital. Following informed consent, the study par‐ with intrapulpal calcification, and gingival overgrowth.1-4 Despite ticipants were given an oral examination with radiographs and oral typically normal blood chemistry, nephrocalcinosis is usually discov‐ photographs. Saliva samples were collected using the Saliva DNA ered by a routine renal ultrasound without subjective complaints but Collection and Preservation kit (Norgen Biotek Corp), and DNA was may not appear until later in life. Among the oral findings, gingival extracted following the manufacturer's instructions. overgrowth is the one that only involves soft tissues and is consis‐ Genomic DNA from the proband was characterized by whole‐ tently found in ERS patients, although its severity varies significantly exome sequencing (Genomics). Briefly, the genomic DNA was cap‐ among reported cases.2,5,6 Histologically, it has been demonstrated tured with SureSelect Human All Exon V5 Kit (Agilent Technologies) that gingival tissues from the patients have well‐structured epithe‐ and sequenced with Illumina HiSeq X Ten for 100 base paired‐end lium with elongated rete ridges and dense connective tissues con‐ reads. Reads were aligned to human reference genome GRCh37 taining an increased amount of collagen fiber bundles running in all (hg19) using BWA. Single‐nucleotide variants and short insertions directions, myofibroblasts, islands of odontogenic epithelium, and and deletions (indels) were called using GATK HaplotypeCaller. The calcified psammomatous deposits (ectopic calcification), indicating called variants were annotated using Ensembl VEP.14 The disease‐ disturbed epithelial and connective tissue homeostasis.5,6 However, causing FAM20A sequence variations were confirmed in the proband the molecular mechanism underlying these abnormalities is largely and his father by Sanger sequencing.4 unknown. Biallelic loss‐of‐function mutations of FAM20A (family with sequence similarity 20, member A; OMIM *611062) were first 2.2 | RNA sequencing and analyses identified to be associated with amelogenesis imperfecta and 7 Gingival tissues were harvested from the proband during surgical gingival fibromatosis syndrome (AIGFS; OMIM #614253) and removal of left maxillary molars and an age‐comparable healthy subsequently found to cause ERS, which differ from AIGFS only 3,4 male patient undergoing a periodontal procedure of distal wedge by the presence of nephrocalcinosis. On the other hand, mu‐ over tooth number 15. Tissues were immediately immerged in 5‐10 tations in FAM20C, the closest paralog to FAM20A, have been volumes of RNAlater® solution (Thermo Fisher Scientific) and in‐ known to cause Raine syndrome (RNS; OMIM #259775) charac‐ cubated overnight at 4°C, and then stored at −20°C until needed terized by deadly osteosclerotic bone dysplasia and intracranial 8 for RNA extraction. Total RNA was extracted from samples and calcifications. Oral manifestations of RNS have been reported checked for integrity using an Agilent 2100 Bioanalyzer with RNA to be similar to those of ERS, including enamel hypoplasia and 6000 Nano Kit. A cDNA library was created by random priming from gingival overgrowth, in some patients with hypomorphic FAM20C 9 fragmented mRNA for synthesis of the first‐strand cDNA followed mutations and a non‐lethal form of RNS. Molecular and struc‐ by PCR amplification. The library was sequenced on an Illumina tural investigations further demonstrated that FAM20C is the NextSeq 500 Sequencer System using 75 bp single‐end sequencing. long‐sought Golgi casein kinase that phosphorylates the vast 10,11 The processed reads were mapped to hg19 human reference tran‐ majority of the secreted phosphoproteome, and FAM20A is a scriptome using bowtie2 (v2.2.6) and selected for following quan‐ pseudokinase that allosterically activates FAM20C to phosphory‐ 15 12,13 tification done by RSEM (RNA Seq by Expectation Maximization). late secreted proteins important for biomineralization. While EBSeq (v1.16.0) was used to identify differentially expressed genes these studies might unravel the molecular mechanisms underlying between ERS and control gingival tissues.16 Further gene ontology enamel defects of ERS, the pathogenesis of gingival overgrowth (GO) and enrichment analyses were conducted using clusterPro‐ and the role of FAM20A in gingival homeostasis remain to be filer (v3.8.1).17 GOplot was implemented to plot the results and to elucidated. perform hierarchical clustering for gene expression patterns (fold In this study, we characterized a case of ERS and reported two changes) or functional categories (GO terms).18 novel FAM20A disease‐causing mutations. By performing RNA se‐ quencing (RNAseq) of gingival tissues from the patient, we char‐ acterized the transcriptome of ERS gingiva and identified aberrant 2.3 | Histological analyses and gene expressions and biological processes that might underlie the immunohistochemistry gingival overgrowth and ectopic calcification in ERS. Histological analyses further validated these findings and suggested potential Gingival tissues harvested from the proband and the con‐ distinct roles of epithelium and connective tissues in altered gingival trol patient were formalin fixed, paraffin embedded, sec‐ homeostasis of ERS. tioned, and H&E stained for regular histological analyses. For WANG et al. | 655 immunohistochemistry, the staining was performed using a 3 | RESULTS VENTANA BenchMark XT automated IHC/ISH staining instru‐ ment (Ventana Medical Systems). Briefly, 4 μm‐thick sections 3.1 | Novel FAM20A mutations causing enamel‐ were deparaffinized, rehydrated, and incubated with 3% hydro‐ renal syndrome gen peroxide solution for 5 minutes. After a washing procedure The proband was a 41‐year‐old male from Taiwan, presumably of East with the supplied buffer, sections were repaired for 40 minutes Asian ethnicity. He came to our clinic for treatment of a progressive with EDTA, followed by incubation with the primary antibody for pain over upper left back teeth. He has been taking gout medication 60 minutes at 37°C and then overnight at 4°C. After three rinses but is otherwise healthy. According to him, his adult teeth came out in buffer, the slides were incubated with the secondary antibody. pretty late and appeared small and widely spaced. His gums have The final staining was visualized with a DAB substrate chromo‐ looked large and bumpy. No other members from his family seemed gen solution from OptiView DAB IHC Detection Kit (Ventana to have teeth that looked like his (Figure 1A). He was diagnosed with Medical Systems). Afterward, slides were counterstained with generalized amelogenesis imperfecta by his family dentist and has hematoxylin, dehydrated, and mounted. The primary antibodies undergone multiple prosthodontic treatments. Clinically, most teeth used in the study included: anti‐FAM20A (1:100; A8496; ABclonal were covered with fixed prostheses, except tooth numbers 2, 14, Technology), anti‐ALPL (1:100; 11187‐1‐AP; Proteintech), anti‐ 15, 29, 31 (Figure 1B). The right maxillary first molar was a dental SPARC (1:100; 15274‐1‐AP; Proteintech), anti‐ACTA2 (1A4; Cell implant. The uncovered teeth showed generally thin dental enamel Marque). The secondary antibodies were from Histofine® Simple with smooth tooth surfaces. Both left maxillary molars exhibited Stain MAX PO (R) (Nichirei Bioscience).

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FIGURE 1 Enamel‐renal syndrome (ERS) family with novel FAM20A mutations. A, Pedigree. Dots mark the two persons who donated samples for DNA sequencing. B, Oral photographs of the proband (III:1). Most teeth were covered with fixed prostheses. The uncovered teeth (tooth numbers 2, 14, 15, 29, 31) show generally thin dental enamel with smooth tooth surface. The attached gingiva is enlarged and bumpy. C, Panoramic radiograph of the proband. Many impacted teeth (tooth numbers 1, 16, 17, 18, 22, 26, 27, 32) show a complete lack of dental enamel and intrapulpal calcifications. Roots of the teeth are generally short. D, Kidney ultrasounds of the proband. Hyperechoic signals are evident and suggestive of medullary nephrocalcinosis. E, DNA sequencing chromatograms of FAM20A mutations. Left: Sequence from the border of Exon 1 and Intron 1 revealing heterozygosity for a one‐nucleotide deletion (g.5417delG; c.129delG) that occurs in the father (II:5) and proband. Right: Exon 5 sequence revealing heterozygosity for a two‐nucleotide deletion (g.62248_62249delAG; c.734_735delAG) and a synonymous SNP (g.62249G > A; rs2286562) that occur in the proband. The mutation designations are with respect to the FAM20A genomic reference sequence NG_029809.1 and cDNA reference sequence NM_017565.3 (for mRNA transcript variant 1) 656 | WANG et al. deep periodontal pockets and moderate tooth mobility. Gingival tis‐ in extracellular matrix biosynthesis, such as COL1A1 (FC = 13.8) and sues appeared slightly thick and firm over the facial surfaces of the COL1A2 (FC = 11.0). Genes related to biomineralization also appeared alveolar ridges. Radiographically, multiple impacted teeth were no‐ to be highly up‐regulated, such as LTF (FC = 1084, the highest in the ticed, including tooth numbers 1, 16, 17, 18, 22, 26, 27, 32 (Figure 1C). rank), SPARC (FC = 6.2), and ALPL (FC = 4.7). On the other hand, many These impacted teeth showed a complete lack of dental enamel and genes involved in keratinocyte differentiation and keratinization, such an evident sign of pre‐eruptive crown resorption without apparent as those of LCE (late cornified envelope) gene family, were among the pericoronal radiolucencies. Pulp chambers appeared generally small most down‐regulated genes (Table S2). with intrapulpal calcifications. Tooth numbers 14 and 15 exhibited Further gene ontology (GO) and enrichment analysis revealed that severe periodontal bone destruction, which might involve impacted 951 GO terms of biological process (bp) were significantly over‐repre‐ tooth number 16. Renal ultrasonographs taken during proband's sented or under‐represented with P‐values ≦ 0.05 (Table S3). To get routine medical examination revealed medullary nephrocalcinosis of an impression if a biological process is more likely to be decreased or bilateral kidneys (Figure 1D). Based upon these findings, a clinical increased, a z‐score was further calculated for each bp GO terms, and diagnosis of enamel‐renal syndrome was made. the first 100 most significant terms were plotted (Figure 2; Table S4). To confirm the clinical diagnosis, exome analysis of pro‐ Z‐score is a simplified value to assess whether the biological process band's DNA was performed. Two deletion mutations of FAM20A is more likely to be decreased (negative value) or increased (positive were identified, g.5417delG (c.129delG; p.Cys44Alafs*101) and value).18 While biological processes involved in extracellular matrix or‐ g.62248_62249delAG (c.734_735delAG; p.Glu245Glyfs*11), ganization (GO:0030198, GO:0043062), angiogenesis (GO:0048514, and subsequently validated with Sanger sequencing (Figure 1E). GO:0001525), and biomineralization (GO:0001501) had the high‐ Proband's father was a heterozygous carrier of p.Cys44Alafs*101 est z‐scores, those related to epithelial development and keratini‐ mutation. However, we were unable to recruit proband's mother, zation (GO:0030216, GO:0031424, GO:0009913, GO:0008544, who should carry the p.Glu245Glyfs*11 mutation. These two mu‐ GO:0043588) had the lowest. Noticeably, while keratinocyte dif‐ tations, located at Exon 1 and 5, respectively, would cause a frame‐ ferentiation (GO:0030216; z‐score = −5.06) is under‐represented, shift followed by a premature stop codon. The mutant transcripts epithelial cell proliferation (GO:0050673; z‐score = 3.78) is over‐rep‐ would presumably undergo nonsense‐mediated decay and produce resented, suggesting a role of FAM20A in regulating epithelial prolif‐ no FAM20A protein. Neither of the two mutations were previously eration and differentiation (Table S4). reported in cases of ERS (Table S1). To further understand the relationships between genes with large fold changes and biological processes of significant enrich‐ ment, 10 biological processes of particular interest were selected 3.2 | Transcriptome analyses of ERS gingival tissues and plotted with the highly up‐regulated and down‐regulated genes To investigate the underlying mechanism of gingival hyperplasia in (Figure 3). Many of these genes were related and functioning in sim‐ ERS, we harvested gingival tissues from the proband during surgical ilar biological processes. Noticeably, genes involved in MAPK, ERK1, removal of left maxillary molars. Transcriptional profiling of the tissues and ERK2 cascades, such as IL6, CCL4, XCL2, CCL8, and NTRK1, was performed using RNAseq and compared with that of control non‐ were mainly over‐expressed in ERS gingivae. In contrast, expressions ERS gingiva. A total of 1683 genes were identified with PPEE ≦ 0.05 of genes that function in proteolysis and keratinocyte differentiation and a minimum fold change (FC) ≧ 2 as being significantly increased or were significantly down‐regulated. These relationships were further decreased in ERS relative to control gingivae (Table S2). Of these, 1129 illustrated by performing hierarchical clustering for gene expression showed higher expression and 554 lower expression in the ERS gin‐ patterns (fold changes) or functional categories (GO terms) to unbi‐ giva. Among highly over‐expressed genes, there were many involved asedly obtain clusters that were likely to contain sets of co‐regulated

FIGURE 2 Barplot of the GO terms of biological process with a significant change. Only the first 100 most significant over‐represented or under‐represented GO terms of biological process are plotted. The y‐axis shows the significance of the terms on a logarithmic scale, and the bars are ordered according to their z‐score (Table S4). The red color indicates a positive z‐score, and the blue color a negative one WANG et al. | 657

FIGURE 3 Circos plot of the relationship between genes with a large fold change (FC) and the biological process (bp) GO terms with significant enrichment. While several highly up‐regulated genes are related to biomineral tissue development (GO:0031214; firebrick), many down‐regulated ones are involved in keratinocyte differentiation (GO:0030216; bisque). Genes within ERK1 and ERK2 cascade (GO:0070371; green‐yellow) appear to be over‐expressed in ERS gingival tissues or functionally related genes (Figure 4). The results were consistent epithelium and a cell‐dense lamina propria (Figure 5A). The epithe‐ with other analyses. lium was mildly acanthotic and had thick broad‐based rete ridges and a thin parakeratinization layer, compared to that of normal gin‐ giva. The connective tissue showed dense collagen fibers running 3.3 | Histological analyses of ERS gingival tissues in various directions with fewer papillae inserted into epithelium. We further performed histological analyses on proband's gingiva Numerous basophilic and laminated calcified structures, resembling to study the histopathology of ERS. The tissue exhibited a normal psammoma bodies, were observed at both superficial and deep architecture of oral mucosa with a well‐structured parakeratinized areas of the connective tissue (Figure 5A,B). Immunohistochemical 658 | WANG et al.

FIGURE 4 Dendrograms of hierarchical clustering for gene expression analyses. Left: Genes are grouped together based on their expression patterns (FC; fold changes). The first ring next to the dendrogram represents the logFC of the genes, whose values are color‐ coded. The next ring represents the terms assigned to the genes from the 10 selected GO terms of biological process, which are also color‐ coded. Right: Genes are grouped based on the functional categories (GO terms) from a functional analysis of the differentially expressed genes performed with clusterProfiler (v3.8.1) analyses against FAM20A on normal gingiva revealed a strong showed a relatively weak reaction in the connective tissue except reactivity at the suprabasal layers of epithelium except the par‐ some moderate staining at blood vessels in the papillae (Figure 6D), akeratinized surface layer (Figure 5D). The positive staining was of which was not observed in proband's tissue. These results of in‐ a dot‐like pattern and localized within the keratinocytes. The nu‐ creased ALPL and SPARC expression in the connective tissue were clei, cell membranes, and extracellular space were negative for the consistent with the findings from transcriptome analyses and in‐ signal. Noticeably, in the connective tissue, the endothelial cells of dicative of an activation of biomineralization‐associated genes in blood vessels and some fibroblasts were also moderately stained. fibroblasts when FAM20A was depleted. On the other hand, α‐SMA In contrast, the FAM20A immunoreactivity on proband's gingiva immunohistochemistry revealed an increased number of blood ves‐ was significantly diminished with only scattered weak staining in sels (α‐SMA‐positive endothelial cells) and myofibroblasts (α‐SMA‐ the epithelium, which confirmed the depletion of the protein due to positive spindle cells) in the connective tissue of proband's gingiva FAM20A null mutations (Figure 5C). compared to the control, which showed barely detectable signals To confirm the enriched biological processes of biomineralization outside of blood vessels (Figure 6E,F). and angiogenesis in ERS gingiva revealed by the transcriptome data, immunostainings of ALPL (Alkaline Phosphatase), SPARC (Secreted Protein, Acidic and Cysteine Rich), and α‐SMA (Alpha Smooth 4 | DISCUSSION Muscle Actin; ACTA2) were conducted. While ALPL weakly stained in the connective tissue of control gingiva, proband's tissues showed In this study, we presented an ERS individual and identified two a moderate to strong ALPL immunoreactivity in fibroblasts and en‐ novel FAM20A frameshift mutations, p.Cys44Alafs*101 and dothelial cells of blood vessels (Figure 6A,B). Particularly, areas sur‐ p.Glu245Glyfs*11. Although these two mutations have never been rounding the psammomatous calcifications were strongly stained reported in ERS kindreds, they are documented in databases of (Figure S1A). For SPARC, a matrix‐associated protein, the positive human variants.19 In gnomAD (Genome Aggregation Database), the staining was detected all over the connective tissue of proband's p.Glu245Glyfs*11 (c.734_735delAG) mutation was identified in 7 out gingiva with the strongest signal around the psammomatous calci‐ of 18 870 chromosomes, giving a MAF (minor allele frequency) of fications (Figure 6C and S1B). On the contrary, the control gingiva 0.000371, in East Asian population (EAS), while p.Cys44AlafsTer101 WANG et al. | 659

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FIGURE 5 Enamel‐renal syndrome (ERS) gingival histology and FAM20A immunohistochemistry. A and B, H&E staining of proband's gingival tissue (100×). A, The epithelium shows mild acanthosis and thick broad‐based rete ridges. Dense collagen fibers running in different direction are seen in the lamina propria with some basophilic calcifications (arrowhead). B, Clusters of psammomatous calcifications (arrowheads) are observed at different areas of gingival connective tissue with no apparent epithelial nests around. C and D, FAM20A localization in gingival tissues from the proband and a control (100×). C, In proband's gingiva, FAM20A immunoreactivity is minimal except some scattered staining in the epithelium. D, In control gingiva, strong FAM20A signals are observed throughout the whole layer of epithelium, except the basal cell layer and the parakeratinized surface layer. Moderate signal is also detected in endothelium of blood vessels and some connective tissue fibroblasts

(c.129delG) was found in one chromosome from EAS in the whole da‐ z‐score), suggesting a regulatory role of FAM20A in balance between tabase. Given that ERS is a recessive disorder, it is expected to identify epithelial proliferation and differentiation. A significant decrease in heterozygous carriers in general population. Of the 41 disease‐caus‐ parakeratinization and mild acanthosis of proband's gingiva on his‐ ing FAM20A mutations reported to date, 14 are documented in either tology are consistent with the transcriptome result. Recently, Li et gnomAD or ExAC (Exome Aggregation Consortium) database (Table al reported that loss of epithelial FAM20A in a conditional knockout S1). These mutations (variants) are considerably rare and mostly iden‐ mouse model (K14‐Cre;Fam20aflox/flox mice) caused a significant thick‐ tified only in a specific population. For example, the p.Leu117Cysfs*22 ening of the gingival epithelium, which further supports our findings (c.349_367del) mutation is found in 3 out of 13 260 chromosomes and the role of FAM20A in epithelial homeostasis.25 On the other in EAS with a MAF of 0.0002262 and has been identified in two hand, aberrations in the connective tissue also contribute to gingival Thai families of ERS, suggesting inheritance from a common ances‐ hyperplasia of ERS. Many biological processes are activated, including tor.5,20 Noticeably, a two‐nucleotide deletion in Exon 1, c.34_35del extracellular matrix (ECM) organization, angiogenesis, and biomineral‐ (p.Leu2Alafs*67), has been reported in 5 families of ERS, but not docu‐ ization (high positive z‐scores), which are respectively corresponding mented in gnomAD or ExAC database, which seems to be a mutation to the dense collagen fibers, abundant blood vessels, and psammo‐ hotspot.3,21-24 However, those cases were all homozygotes for the matous calcifications on histopathology of ERS gingiva. While many mutation and mostly from Mediterranean region with probably similar genes involved in collagen metabolic processes and fibril organization genetic background, indicating a potential founder effect. Therefore, are over‐expressed in the connective tissue, expression of genes regu‐ identity by descent (IBD) seems to underlie the genetic etiology of lating proteolysis is significantly affected, demonstrating a modulating many ERS cases reported all over the world. role for FAM20A in ECM homeostasis. Showing FAM20A expression Gingival hyperplasia has been considered as one of the character‐ in endothelial cells of blood vessels in normal gingiva and increased istic features of ERS, although the severity of individual cases can vary blood vessel formation in ERS gingiva, we revealed potential unap‐ significantly.2 Based upon our transcriptome and histological analyses preciated functions of FAM20A in angiogenesis. However, although of ERS gingiva, this pathology seems attributable to aberrations of α‐SMA, an endothelial marker, is over‐expressed in the gingiva when both epithelium and underlying connective tissues. In ERS epithelium, FAM20A is depleted, the increased expression seems to result from while cell proliferation is activated (positive z‐score), keratinocyte dif‐ an increased number of myofibroblasts which also express α‐SMA ferentiation and keratinization are significantly suppressed (negative in addition to endothelial cells of blood vessels. The functions of 660 | WANG et al.

(A) (B) FIGURE 6 ALPL, SPARC, and α‐SMA immunohistochemistry. A and B, ALPL immunostaining in gingival tissues from the proband and a control (100×). A, In proband's gingiva, immunoreactivity is strongly detected in the connective tissue fibroblasts. B, In control gingiva, the reactivity is scattered and weak. C and D, SPARC immunostaining in gingival tissues from the proband and a control (100×). C, In proband's gingiva, signals are strong in the connective tissue fibroblasts (C) (D) but weak in blood vessel endothelium. No signals show in the epithelium. D, In control gingiva, signals are generally weak, except some strong staining in endothelial cells of blood vessels at connective tissue papillae (arrowhead). E and F, α‐SMA immunostaining in gingival tissues from the proband and a control (200×). E, In proband's gingiva, reaction is strong in both the connective tissue (myofibroblasts) and blood vessel endothelium. F, In control gingiva, only (E) (F) smooth muscle cells on vessel walls and endothelial cells show strong signals

FAM20A in angiogenesis and myofibroblasts differentiation require and enhances extracellular protein phosphorylation within the further investigations. Ectopic gingival calcifications have been con‐ secretory pathway.12,13 However, how loss of this function leads sistently observed in ERS patients.2,5,6 It has also been suggested that to all the pathologies in ERS remains puzzling. An alternative ex‐ odontogenic epithelial cells were responsible for formation of these planation is that the ERS phenotypes might result from loss of calcifications due to their close proximity to the nests of epithelial unidentified G‐CK‐independent functions of FAM20A. However, cells.6 However, there are few, if any, epithelial nests observed around this is unlikely, since FAM20C loss‐of‐function mutations cause the psammoma bodies in our case. Also, genes involved in biomin‐ similar and overlapping phenotypes of ERS, including gingival hy‐ eralization, such as ALPL and SPARC, are over‐expressed in the con‐ perplasia.9,26,27 As discussed above, depletion of FAM20A might nective tissue surrounding the calcified structures, suggesting that a promote proliferation, while inhibiting differentiation, in gingival pathological calcification process is activated in connective tissue fi‐ epithelium. Disturbance in this proliferation/differentiation bal‐ broblasts when FAM20A is depleted. The lack of gingival calcifications ance probably results from impaired phosphorylation of secreted in Fam20a epithelial conditional knockout mice further supports this signaling molecules, such as EGF, since many of these proteins hypothesis.25 Moreover, this aberrant mineralization is probably not contain G‐CK phosphorylation motif.11 Although diminished phos‐ due to a disturbance in systemic calcium homeostasis, since the blood phorylation of enamel matrix proteins is considered as the main chemistry is usually normal in ERS patients,2 including our proband. cause of enamel defects in ERS,12,28 aberrant epithelial prolifer‐ Therefore, localized activation of biomineralization‐related genes in ation/differentiation balance might also partly contributes to the connective tissue fibroblasts, when FAM20A is depleted, seems to pathogenesis. It has been shown that ameloblast differentiation at underlie gingival ectopic calcification in ERS. This mechanism might pre‐secretory stage, prior to secretion of enamel matrix proteins, also apply to parenchymal calcification in kidneys (nephrocalcinosis) was disturbed in Fam20a knockout mice, which supports this hy‐ of ERS patients. pothesis.25,29 Like epithelial abnormalities, loss of FAM20A's func‐ Recently, FAM20A was shown to be a pseudokinase that forms tion in facilitating phosphorylation of secreted signaling molecules a functional complex with Golgi casein kinase (G‐CK) Fam20C by G‐CK might also underlie the pathology in the connective tissue, WANG et al. | 661 including excessive accumulation of extracellular matrix and ec‐ 13. Cui J, Zhu Q, Zhang H, et al. Structure of Fam20A reveals a pseu‐ topic calcifications. Comprehensive characterization and analysis dokinase featuring a unique disulfide pattern and inverted ATP‐ binding. eLife. 2017;6:e23990. of “phospho‐secretome” from diseased tissues will be required to 14. McLaren W, Gil L, Hunt SE, et al. The ensembl variant effect predic‐ test the hypothesis and unravel the key affected phosphoproteins tor. Genome Biol. 2016;17:122. driving the various pathologies of ERS. 15. Li B, Dewey CN. RSEM: accurate transcript quantification from RNA‐Seq data with or without a reference genome. BMC Bioinformatics. 2011;12:323. ACKNOWLEGEMENTS 16. Leng N, Dawson JA, Thomson JA, et al. EBSeq: an empirical Bayes hierarchical model for inference in RNA‐seq experiments. We thank the study participants for their contributions and Dr Bioinformatics. 2013;29:1035‐1043. Jia‐Huey Hwang for providing dental records. This work was sup‐ 17. Yu G, Wang LG, Han Y, He QY. clusterProfiler: an R package ported by National Taiwan University Hospital (NTUH) (Grant 106‐ for comparing biological themes among gene clusters. OMICS. 2012;16:284‐287. N3424); the Ministry of Science and Technology in Taiwan (MOST) 18. Walter W, Sanchez‐Cabo F, Ricote M. 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