PLoS ONEplosplosonePLOS ONE1932-6203Public Library of ScienceSan Francisco, CA USA10.1371/journal.pone.0227287PONE-D-19-23967Research ArticleBiology and life sciencesPhysiologyDigestive physiologyDentitionMedicine and health sciencesPhysiologyDigestive physiologyDentitionPhysical sciencesChemistryPhysical chemistryChemical bondingHydrogen bondingBiology and life sciencesBiochemistryProteinsProtein domainsHomeoboxBiology and life sciencesGeneticsMutationFrameshift mutationBiology and life sciencesAnatomyDigestive systemTeethMedicine and health sciencesAnatomyDigestive systemTeethBiology and life sciencesAnatomyHeadJawTeethMedicine and health sciencesAnatomyHeadJawTeethBiology and life sciencesGeneticsMutationMissense mutationBiology and life sciencesGeneticsMutationInsertion mutationResearch and analysis methodsDatabase and informatics methodsBiological databasesMutation databasesBiology and life sciencesGeneticsMutationMutation databasesTwo novel mutations in MSX1 causing oligodontiaMSX1 mutations causing oligodontiaYangLeConceptualizationData curationSoftwareSupervisionVisualizationWriting – original draftLiangJiaFunding acquisitionInvestigationMethodologyProject administrationResourcesSoftwareValidationVisualizationWriting – original draftWriting – review & editingYueHaitangInvestigationMethodologyProject administrationhttp://orcid.org/0000-0002-8035-4276BianZhuanConceptualizationData curationFormal analysisSupervisionValidationVisualizationWriting – review & editing*The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, Hubei Province, ChinaCaiTaoEditorNIDCR/NIH, UNITED STATES
The authors have declared that no competing interests exist.
* E-mail: bianzhuan@whu.edu.cn8120202020151e0227287892019161220192020Yang et alThis is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Tooth agenesis is one of the most common developmental anomalies in humans and can affect dental occlusion and speech pronunciation. Research has identified an association between mutations in MSX1, PAX9, EDA, AXIN2, WNT10A, WNT10B and LRP6 and human tooth agenesis. Two unrelated individuals with non-syndromic tooth agenesis and their families were enrolled in this study. Using Sanger sequencing of the candidate genes, we identified two novel mutations: a missense mutation c.572 T>C and a frameshift mutation c.590_594 dup TGTCC, which were both detected in the homeodomain of MSX1. After identifying the mutations, structural modeling and bioinformatics analysis were used to predict the resulting conformational changes in the MSX1 homeodomain. Combined with 3D-structural analysis of other MSX1 mutations, we propose that there is a correlation between the observed phenotypes and alterations in hydrogen bond formation, thereby potentially affecting protein binding.
National Key Research and Development Program of China2016YFC1000505http://orcid.org/0000-0002-8035-4276BianZhuanKey Research Program of Provincial Department of Science and Technology2017ACA181http://orcid.org/0000-0002-8035-4276BianZhuanhttp://dx.doi.org/10.13039/501100003819Natural Science Foundation of Hubei Province2017CFB212LiangJiaThis study was supported by grants from the General Program of National Natural Scientific Foundation of China (No. 81970923), National Key Research and Development Program of China (No. 2016YFC1000505), Key Research Program of Provincial Department of Science and Technology (No. 2017ACA181), and Natural Science Foundation of Hubei Province (No. 2017CFB212).Data AvailabilityAll relevant data are within the paper and its Supporting Information files.Introduction
Tooth agenesis is a common developmental anomaly of the human dentition. In the general population, the incidence of tooth agenesis of permanent teeth ranges from 2.2% to 10.1% [1]. Several terms are used to describe tooth agenesis: hypodontia is referred as an absence of less than six teeth excluding the third molars; oligodontia is an absence of six or more teeth excluding the third molars; and anodontia is the complete absence of teeth [2]. Tooth agenesis is classified as syndromic when it is accompanied by other inherited abnormalities including nail dysplasia [3], sparse hair, lack of sweat glands or a cleft lip and palate [4–8], and classified as non-syndromic when the absence of teeth is an isolated characteristic. Tooth agenesis occurs either in a sporadic or in a hereditary manner, in autosomal dominant [9], autosomal recessive [10] or X-linked patterns [11]. The etiology of tooth agenesis is complex and not yet completely elucidated [2]. Environmental factors potentially influencing the dental development include trauma, chemotherapeutic drugs, radiotherapy or thalidomide use during pregnancy [12, 13]. The majority of cases are caused by genetic mutations, and to date, mutations in the MSX1, PAX9, AXIN2, WNT10A, EDA, EDAR, EDARADD, WNT10B and LRP6 genes have been associated with non-syndromic tooth agenesis (NSTA) cases [14–22].
MSX1 and PAX9 are the first genes identified causing NSTA [17, 19]. Both genes are transcription factors that play crucial part during the bud to cap stages of odontogenic development. Msx1 and Pax9 can interact with each other and they also act synergistically to activate the Bmp4 which is critical for tooth development [23]. EDA, EDAR and EDARADD are candidate genes of both NSTA and STA. EDA/EDAR/EDARADD signaling has been shown to play an important role in NSTA [24]. Recently, more and more genes in Wnt signaling pathway have been found to be related to NSTA. The first gene of Wnt signaling pathway involved in NSTA was AXIN2 [20]. This gene was also found to be associated with a variety of cancers [25]. Axin2 plays a critical role in regulating the stability of β-catenin in the Wnt signaling pathway [26]. The second gene was WNT10A which encodes a secreted signaling protein. WNT10A mutations have been reported to be linked to a majority of NSTA cases [16]. Wnt10a is a key mediator of Wnt signaling which is required for proper tooth development [27]. In 2015, mutations in the WNT co-receptor LRP6 were identified in families with autosomal-dominant non-syndromic oligodontia [15]. In 2016, WNT10B mutations causing oligodontia had been reported in a Chinese population [14].
Among those genes, NSTA is mostly associated with mutations in PAX9, MSX1, WNT10A, AXIN2 and EDA [28–30]. Therefore, in this study, we searched for mutations in the above five genes in two unrelated individuals with non-syndromic oligodontia. The study objectives were: 1) to identify the mutations responsible for the tooth agenesis in two Chinese patients; 2) to use 3D-structure modeling to explain the reasons why these mutations can lead to tooth agenesis.
Materials and methodsSubjects
Two unrelated Chinese oligodontia patients were identified in the orthodontic department at the School of Stomatology, Wuhan University. Intraoral examination and panoramic radiographs were taken to verify the number and location of missing teeth of each patient. Pedigree construction was achieved using clinical examinations and verbal interviews with the available family members. Blood samples were collected from the probands, their available family members and 300 unrelated healthy controls. All procedures were approved by the National Natural Science Foundation of the Medical Ethics Committee of the Stomatological Hospital of Wuhan University, China (Ethics Approval Identifier: A 42, date of approval: March 4, 2019). All participants in this study gave their written informed consent to publish these case details.
Identification of mutations
Genomic DNA was extracted from the peripheral blood samples of all consenting family members and controls according to standard salt extraction procedures [31]. Screening of pathogenic mutations was performed using polymerase chain reaction (PCR) amplification and sequencing the complete exons and exon–intron boundaries of these five genes: MSX1 (ENSG00000163132), PAX9 (ENSG00000198807), EDA (ENSG00000158813), AXIN2 (ENSG00000168646) and WNT10A (ENSG00000135925).
Multiple sequence alignments
Multi-species amino acid sequence alignment of the MSX1 protein sequence (NP_002439.2) was performed using Clustal X (https://www.ebi.ac.uk/Tools/msa/). MSX1 sequences from dolphin to human were obtained from ENSEMBL.
3D modeling of MSX1 variants
On the basis of the crystal structure of the MSX1 homeodomain complex with DNA [32], the three-dimensional structure of the wild-type MSX1 homeodomain was derived using SWISS-MODEL (https://www.swissmodel.expasy.org) with PDB: 1ig7.1.C as a template. The 3D model was constructed using Swiss Pdb Viewer V 4.1 [33]. Visualization of the three-dimensional (3D) structures was performed with PyMOL (The PyMOL Molecular Graphics System, Version 0.99rc6, Schrödinger, LLC., Cambridge, MA, USA).
Statistical analysis
Differences between the average number of missing teeth caused by missense mutations in the MSX1 homeodomain with or without alterations in hydrogen bonding were analyzed using an unpaired, two-tailed t-test with GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA, USA). P-value < 0.05 was considered significant.
ResultsPedigree and phenotype analysis
Pedigree analysis of the two families revealed that the proband (Ⅱ2) in family 1 was a sporadic patient and oligodontia in family 2 was exhibited in an autosomal dominant manner (Fig 1A). The proband (Ⅱ2) in family 1 was a 16-year-old female with the congenital absence of ten permanent teeth excluding third molars. The second upper deciduous teeth were retained (Fig 1B and 1C), and both of her parents and her elder brother had normal dentition. The proband (Ⅲ1) in family 2 was a 21-year-old male with more severe tooth agenesis, characterized by the absence of twenty permanent teeth excluding the third molars. The shape of 25 was conic, and most deciduous teeth were retained (Fig 1B and 1C). The mother of the proband (Ⅱ2) was also diagnosed as tooth agenesis through oral examination. However, she could not recall or confirm extraction of some teeth. No orofacial cleft or other craniofacial abnormalities were noted in the affected members of the two families. Additionally, all reported subjects had normal primary dentition, nails, skin, and hair.
10.1371/journal.pone.0227287.g001
Identification of two Chinese families with non-syndromic tooth agenesis.
(A) Pedigrees. Filled and unfilled symbols indicate affected and unaffected individuals, respectively. Squares and circles represent males and females, respectively. (B) Panoramic radiograph of the probands. The white stars indicate missing permanent teeth. Left: The proband of family I. Right: The proband of family II. (C) Tooth phenotypes of the probands with oligodontia. Stars indicate congenital missing tooth and triangle denotes cone-shaped teeth.
Mutation analysis
Mutation analysis of candidate genes detected two novel heterozygous mutations in MSX1 in two separate families. The proband of family 1 demonstrated a missense mutation at c.572T>C (Fig 2A). Her parents were wild-type at c.572, indicating a de novo mutation. A cross-species alignment of the MSX1 protein showed that p. Phe191 is highly conserved in the homeodomain (Fig 2B). This mutation replaced the hydrophobic phenylalanine with a hydrophilic serine at amino acid position 191. Screening of candidate genes in the proband of family 2 showed a frameshift insertion c.590_594 dup TGTCC in MSX1 (Fig 2C). His mother also carried the mutation. This mutation is occurred in the homeodomain of MSX1, introducing 20 novel amino acids following the first 198 amino acids, while deleting the normal protein sequence from Ile199 through The303. Neither mutation was detected in the control group and had not been reported by 1000 Genomes, dbSNP, Human Gene Mutation Database (professional version) or PubMed. No pathogenic mutations were found in the PAX9, AXIN2, EDA and WNT10A genes of these two patients.
10.1371/journal.pone.0227287.g002
MSX1 mutations isolated from tooth agenesis patients.
(A) DNA sequence chromatograms presenting a de novo heterozygous missense mutation c.572T>G in MSX1 identified in family I, compared with wild-type control. (B) Conservation analysis shows that the Phe residue at 191 in Msx1 is conserved across human, gorilla, rhesus, mouse, horse, bovin, pteropus alecto, dophin, pteropus vampyrus. (C) DNA sequence chromatograms showing a heterozygous c.590_594 dup TGTCC mutation identified in family II, compared with wild-type.
Bioinformatics analyses
Secondary structural analysis showed that the homeodomain of the wild-type MSX1 protein consisting of 60 amino acids (No.172-231) is composed of the N-terminal arm (NT arm, No.172-180) and three α-helices: helix I (No.181-193), helix II (No.199-209) and helix III (No.213-231). The mutation p. Phe191Ser was located at the end of the helix I (Fig 3A and 3B). In the wild-type MSX1 protein, the phenyl ring of the p. Phe191 was larger and overlapped with p. Gln195 (Fig 3A). In the p. Phe191Ser-mutated MSX1 protein, the phenyl ring disappeared and resulted in a large gap between p.191Ser and p. Gln195 (Fig 3B). Therefore, the p.Phe191Ser mutation may result in an abnormal structure of the MSX1 protein. In the wild type homeodomain, modelling predicted one hydrogen bond between Phe191 and Leu187 (Fig 3C), while in the mutated protein, there are two additional hydrogen bonds to Glu188 (Fig 3D).
10.1371/journal.pone.0227287.g003
Structural modeling of the wide-type and the mutated homeodomain of MSX1 protein.
(A, B) Structural modeling of the wide-type and the p. Phe191Ser mutated homeodomain of MSX1 protein show that the mutation is located at the end of the helix I. (A) In the wild-type MSX1 protein, the phenyl ring of the p. Phe191 is large and overlapped with p. Gln195. (B) In the p. Phe191Ser-mutated MSX1 protein, the phenyl ring disappears, a large gap between p.191Ser and p.Gln195 arise. (C) Phe191 forms a hydrogen bond with Leu187. (D) The Phe191Ser mutation creates another two new hydrogen bonds with Glu188. (E) Structural modeling of the c.590_594 dup TGTCC mutated homeodomain of MSX1protein by Pymol. The mutant structural shows that helix I is conserved, but helixes II and III are disrupted.
The frameshift insertion (c.590_594 dup TGTCC, p.L197SfsX22) is located at the end of the loop domain between helix I and II, and generates a premature stop codon after an unrelated polypeptide sequence consisting of 22 amino acid residues. The shortened mutant-MSX1 protein is composed of two α-helices: helix I (No.181-193), and helix II (No.200-204), only the first helical region (helix I) was conserved and the other two helical regions (helixes II and III) are disrupted (Fig 3E).
Discussion
MSX1 is the first gene identified causing NSTA, which encodes a transcription factor [19]. It is widely expressed in many organs, particularly during the bud and cap stages of tooth development where epithelial–mesenchymal interactions occur in odontogenesis [34]. MSX1 consists of two exons, the second of which includes the highly conserved homeodomain (HD). The homeodomain is comprised of an extended N-terminal arm and three α-helices [35]. The residues in helices I and II are thought to play an important role in structural stability and binding activity while the residues in helix III combined with those in the N-terminal arm are important for DNA binding specificity[36]. It has been demonstrated that the homeodomain is important for the successful interaction of MSX1 with other proteins such as TATA box binding protein (TBP) and distal-less homeobox (DLX) [37, 38]. However, the role of MSX1 in human craniofacial and dental development has not been fully elucidated [39].
As we know, proteins fold into specific configurations mainly through a large number of non-covalent interactions, including hydrogen bonds, ionic bonds, van der Waals forces and hydrophobic interactions [40]. Hydrogen bonding plays a critical role in secondary structure formation and the integrity of three-dimensional structures [41]. In the homeodomain of MSX1, the stable secondary helical structure is maintained by hydrogen bonds between C = O and N-H groups of different amino acids. In this study, we show that the missense mutation Phe191Ser may lead to two additional hydrogen bonds with Glu188. Moreover, the mutated side chain becomes smaller, resulting in an increased distance between p.191Ser and p.Gln195. This alteration may force the neighboring residues to rearrange their positions, compromising the stability of the homeodomain and affecting the binding ability of MSX1 to DNA.
In order to elucidate the relationship between the hydrogen bond modifications induced by MSX1 mutations and hypodontia phenotypes, we performed homology modeling to analyze the distribution of mutation sites in the homeodomain. We included 7 MSX1 missense mutations from all missense/nonsense MSX1 mutations in HGMD (Human Gene Mutation Database, professional version 2019.3) [42], which are believed to cause non-syndromic tooth agenesis (Table 1)(S2 Table) (S2 Fig). Large deletions and insertions, small in-frame deletions, and nonsense mutations were not included because these mutations would damage the overall structure of MSX1 protein.
10.1371/journal.pone.0227287.t001
MSX1 homeodomain missense variants in subphenotypes of increasing severity of non-syndromic tooth agenesis (NSTA) and alterations in hydrogen bonds.
MSX1variants(HGVS; cDNA)
MSX1variants(HGVS; predicted protein)
Missing teeth no.
Average no. of missing teeth
No. of patients
Phenotype
Alterations in hydrogen bonds
location
Reference
c.539C>T
p.(T180I)
5
5
1
Sporadic form of hypodontia
No
NT arm
[43]
c.689T>C
p.(L230P)
2–4
3.6
5
Autosomal dominant, non-syndromic hypodontia
No
Loop 3
[44]
c.572 T>C
P.(F191S)
10
10
1
Sporadic form of oligodontia
Add
Helix I
Thisstudy
c.605G>C
p.(R202P)
4–11
6.8
9
Autosomal dominanttooth agenesis
Reduce
Helix II
[19]
c.632T>G
p.(L211R)
8–18
11.7
3
Autosomal dominant, non-syndromic oligodontia
Add
Loop 2
[43]
c.673G>Ac
p.(A225T)
11–19
15
2
Autosomal recessive oligodontia with dental anomalies
Add+Reduce
Helix III
[45]
c.680C>A
p.(A227E)
5–13
8
4
Autosomal dominant, non-syndromic, oligodontia
Add
Helix III
[46]
Our structural modeling predicts a correlation between observed phenotypes and the hydrogen bond alterations. MSX1 mutations that altered hydrogen bonding tend to cause a more severe NSTA phenotype with more missing teeth (the average number of missing teeth were 3.8 vs. 8.8) (S3 Table) (Fig 4). Although hydrogen bonds are not the tractive force of protein or peptide folding, these bonds significantly contribute to the maintenance of the peptide folding [47]. Missense mutations that affect hydrogen bonds may have a significant effect on protein stability, DNA binding specificity, protein expression and interactions. Therefore, it is likely that the mutations we report in this study resulted in more severe selective tooth agenesis. Further functional analysis of these mutations will help to reveal the molecular mechanism of their action.
10.1371/journal.pone.0227287.g004
Statistical analysis.
Comparison of the average missing teeth number caused by missense mutations in MSX1 homeodomain with and without hydrogen bonds alteration obtained from different non-syndromic tooth agenesis patients (patients caused by missense mutations that had no hydrogen bond alteration, n = 6, vs. patients caused by missense mutations that had hydrogen bonds alteration, n = 19). Data are shown as mean ± SEM. An unpaired t test with two-tailed p-values was performed using GraphPad Prism 5 software. P value = 0.0086 obtained by analyzing data. **denotes a P-value <0.01.
In our study, a novel frameshift insertion of 5 bp (TGTCC) in the homeodomain of the MSX1 gene (NM_002448) was also identified in a Chinese family with autosomal dominant non-syndromic tooth agenesis, affecting the amino acid sequence of the homeodomain at p. 197Leu, changing the amino acid sequence from position 199. This alteration changes the spiral shape of helix II and eliminates the helix III, which is important for DNA-binding and protein-protein interactions including the TBP and DLX families [48]. Previous studies have shown that consensus residues that interact with DNA are located in helix III and the N-terminal arm [35]. We speculate that this novel frameshift mutation has a significant impact on the structure of the truncated protein, leading to severe consequences in protein interactions and DNA binding.
This frameshift mutation likely has considerable significant downstream effects as it demonstrates a substantial impact on primary protein structure. The MSX1 homeodomain contacts its target DNA regions in the major and minor groove at helix III and the N-terminal arm, respectively. The insertion variant found in the present study is predicted to be harmful through structural modeling and bioinformatics analyses, and is predicted to affect the DNA-binding functions of the homeodomain. Further studies are required to investigate the subcellular localization of this mutant MSX1 protein in vitro, to verify whether this is a functionally null variant.
Conclusions
In our current study, we described two novel variants of the MSX1 gene identified in two Chinese patients with isolated tooth agenesis: c.572T>C and c.590_594 dup TGTCC. These two novel mutations were proven to be pathogenic with respect to amino acid structure. These results expanded the mutational spectrum of the MSX1 gene and the options to be considered for their precision treatment later. Further expression and functional studies are required to assess the effect of the identified mutations at the protein level.
Supporting information
Comparison of wild type and c. 590_594 dup TGTCC protein sequences of the MSX1 homeodomain underlined.
(TIF)
Predicted mutational impacts on the MSX1 homeodomain structure.
Thr180Ile and Leu230Pro all caused less than 6 missing teeth, neither mutation seems to cause any obvious changes in the structural interface, possibly explaining the milder effects. The other mutations all caused more than 6 missing teeth and had alterations in hydrogen bond formation. The alterations may lead to changes of the helical conformation, ultimately resulting in an alteration of protein folding and decreased stability.
(A) Locations of the mutational sites on the structural model of human MSX1 homeodomain structure (MSX1 homeodomain green, mutational sites red).
(B)- (G) Pair-wise comparisons between the wild-type (left) and mutant (right) residues for predicted changes in local contacts with other amino acids. The salmon stick models indicate wild-type residues, yellow indicate mutated residues, gray indicate neighboring residues, with the short bar in white, red and blue standing for the carbon, oxygen and nitrogen atoms, while the dashed lines in red and blue illustrated the hydrogen bonds. Here, we define a hydrogen bond geometrically as having a donor–acceptor distance ≤3.3 Å. The hydrogen bond distances are determined by Swiss-pdb Viewer.
(B) Leu230 and the Leu230Pro mutation both do not have hydrogen bond interactions with adjacent residues. So, the mutation does not seem to cause any obvious changes in the structural interface, possibly explaining the milder effects found for this mutation.
(C) Thr180 is predicted to have hydrogen-bonding interactions with Gln183 and Leu184. The Thr180Ile mutation does not alter hydrogen-bonding with these two amino acids.
(D) Arg202, located in the α helix II, forms hydrogen bonds with adjacent residues Ser206 located in the same α helix II and Ser198 in the loop and Glu213 in the α helix III. The p. Arg202Pro mutation is predicted to abolish the hydrogen bonding with Ser198 and Glu213.
(E) Leu211 is predicted to have hydrogen-bonding interaction with Ser206 located in the same α helix II. The mutant protein has a longer side chain than the wild-type protein. The Leu211Arg introduce two new hydrogen bonds with Ser210 in the loop.
(F) Ala225 forms hydrogen bonds with adjacent residues Gln221, Asn222 and Arg229, The p. Ala225Thr mutation appears to eliminate the hydrogen bond with Asn222, but creates a new hydrogen bond with Gln221 in the same a-helix.
(G) Ala227 forms hydrogen bonds with adjacent residues Arg223 and Arg224, the p.Ala227Glu mutation appears not to affect these hydrogen bonds but introduce two new hydrogen bonds with Arg223 and Leu230.
The figures are prepared using Pymol.
(TIF)
Primers for candidate genes.
(DOCX)
Missense /nonsense MSX1 mutations from HGMD (2019.3).
(DOCX)
The (average) number of patients and missing teeth in MSX1 mutations with and without hydrogen-bonding alteration.
(DOCX)
The authors of this study would like to thank the patients and their families for their important contribution to this study.
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Novel MSX1 mutation in a family with autosomal-dominant hypodontia of second premolars and third molars. . 2012;57(6):790–5. Epub 2012/02/03. doi: 10.1016/j.archoralbio.2012.01.00322297032.ChishtiMS, MuhammadD, HaiderM, AhmadW. A novel missense mutation in MSX1 underlies autosomal recessive oligodontia with associated dental anomalies in Pakistani families. . 2006;51(10):872–8. Epub 2006/08/26. doi: 10.1007/s10038-006-0037-x16932841.XuanK, JinF, LiuYL, YuanLT, WenLY, YangFS, et al. Identification of a novel missense mutation of MSX1 gene in Chinese family with autosomal-dominant oligodontia. . 2008;53(8):773–9. Epub 2008/04/01. doi: 10.1016/j.archoralbio.2008.02.01218374898.ScheikeJA, BaldaufC, SpenglerJ, AlbericioF, PisabarroMT, KokschB. Amide-to-ester substitution in coiled coils: the effect of removing hydrogen bonds on protein structure. . 2007;46(41):7766–9. doi: 10.1002/anie.20070221817876795.ZhangH, HuG, WangH, SciavolinoP, IlerN, ShenMM, et al. 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14 Oct 2019
PONE-D-19-23967
Two Novel Mutations in MSX1 Causing Oligodontia
PLOS ONE
Dear Dr. Bian,
Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process by two reviewer's comments and suggestions.
This editor has additional suggestions:
In abstract: page 2, line 12: it seems the “with” should be replaced with “between”;
In abstract: page 2, line 22: “ultimately” can be replaced with “thereby potentially”;
In the Introduction part, 2nd paragraph (dealing with the MSX1 gene) could be moved to Discussion part. Instead, briefly introducing signaling and genetic contributions of each of nine genes (mentioned in the 1st paragraph) to tooth agenesis.
In the Introduction part, 3rd paragraph: please give reasons why the five genes (MSX1, et al.) are selected to screen the affected individuals.
In Results part, page 8, line 141: please add the range of homeodomain in amino acid residue numbers (from amino acid residual 172 to 234?).
Based on current version of HGMD, the number of different missense mutations in the HOX domain should be at least 12, not 7.
Page 10, line 186-196, figure 3C-3D of this part can be included as a supplementary figure.
In Discussion part, page 11, line 205-206, replace “demonstrate” with “show” and “leads” with “may lead”.
Page 11, line 211-212, the statement in 2016 may not be true any longer. Please check current professional version of HGMD.
Page 11, line 215-220, again, the number missense mutations in the HOX domain is more than 7. Please check current professional version of HGMD, and try to include all missense alleles.
Figure 4 can be used as supplementary figure.
Suggestion: Please consider to have a phenotypic comparison of all missense-related in non-HOX domain to the Table 3 results. If it turns out to be significant, Table 2 as a summary can be added to the main-text.
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Reviewer #1: Yes
Reviewer #2: Partly
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Reviewer #1: N/A
Reviewer #2: No
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Reviewer #1: Yes
Reviewer #2: Yes
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Reviewer #1: Yes
Reviewer #2: Yes
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Reviewer #1: 1. In this study, authors identified the MSX1mutations responsible for the tooth agenesis in two Chinese patients, and explained the reasons why these mutations could lead to tooth agenesis by 3D-structure modeling. On the other hand, the authors reviewed all known MSX1 missense mutations available as that are believed to cause non-syndromic tooth agenesis. So I suggest that the title should be changed to “Two Novel Mutations in MSX1 Causing Oligodontia: Case Report and Literature Review”.
2. A frameshift mutation c.590_594 dup TGTCC was detected in family 2. In order to be more accurate, cloning and sequencing should be used to detect the frameshift mutation in III:1 and II:2 in family 2.
Reviewer #2: The manuscript by Yang et al reported two novel MSX1 mutations responsible for non-syndromic tooth agenesis. The authors also reviewed genotype-phenotype relationship of MSX1 missense mutations via 3D-structural analysis and concluded that MSX1 mutations that altered hydrogen bonding tend to cause a more severe non-syndromic TA phenotype with more missing teeth. The analysis is based on 7 mutations identified in the published literature. Overall the study is interesting and well designed. However, there are some issues that need to be addressed before publication.
1. The author concluded that MSX1 mutations that altered hydrogen bonding tend to cause a more severe non-syndromic TA phenotype with more missing teeth. How did they get this conclusion? Because there was not any statistical genotype-phenotype correlation analysis compared between the MSX1 mutations with or without altered hydrogen bonding. What is the average number of missing teeth in MSX1 mutations with or without altered hydrogen bonding respectively? How many MSX1 mutations without altered hydrogen bonding were included for the statistical analysis and the reason for selection should be well described.
2. The manuscript needs to be carefully revised and well organized. In the Discussion part, from line 211 to line 282, include the descriptions of methods, results and discussion about MSX1 mutations that altered hydrogen bonding seems to cause more severe non-syndromic TA phenotype.
3. All the figures are of poor quality, not clear. They should provide better and higher resolution images to readers.
4. Mutational analysis was only performed in the proband of family 2. The pathogenic mutations of other members in this family are needed to be confirmed, especially his mother (II:2),since she was also a tooth agenesis patient.
5. How did they get the results of Figure 5 should be well descripted, because that is the key result supporting their conclusion. It should be include in the main body of the manuscript.
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Reviewer #1: No
Reviewer #2: No
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Submitted filename: Comments on PONE-D-19-23967.docx
10.1371/journal.pone.0227287.r002Author response to Decision Letter 0Submission Version1
3 Dec 2019
We have revised the parts of the manuscript you proposed to be well organized. The details can be found in "response to reviewers".
Submitted filename: Response letter.docx
10.1371/journal.pone.0227287.r003Decision Letter 1CaiTaoAcademic Editor2020Tao CaiThis is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Submission Version1
17 Dec 2019
Two Novel Mutations in MSX1 Causing Oligodontia
PONE-D-19-23967R1
Dear Dr. Bian,
We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.
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With kind regards,
Tao Cai, M.D., Ph.D.
Academic Editor
PLOS ONE
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Reviewer #2: All comments have been addressed
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2. Is the manuscript technically sound, and do the data support the conclusions?
The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.
Reviewer #2: Yes
**********
3. Has the statistical analysis been performed appropriately and rigorously?
Reviewer #2: Yes
**********
4. Have the authors made all data underlying the findings in their manuscript fully available?
The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.
Reviewer #2: Yes
**********
5. Is the manuscript presented in an intelligible fashion and written in standard English?
PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.
Reviewer #2: Yes
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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)
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Reviewer #2: No
10.1371/journal.pone.0227287.r004Acceptance letterCaiTaoAcademic Editor2020Tao CaiThis is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
27 Dec 2019
PONE-D-19-23967R1
Two Novel Mutations in MSX1 Causing Oligodontia
Dear Dr. Bian:
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