Association between LTF/MMP20/CA6/TAS1R2 polymorphisms and susceptibility to dental caries.

OBJECTIVES: The aim was to assess the associations between the LTF, MMP20, CA6, and TAS1R2 polymorphisms and caries in the Zhuang population and explore the underlying mechanism of the impact of lactoferrin on caries susceptibility. METHODS: A case-control study of 315 adolescents was conducted in Guangxi, China, from May-November 2022. Data were collected through oral examinations and questionnaires. Buccal mucosa cells and DNA samples were collected using the SNPscan technique. Saliva and supragingival plaque samples were taken from 69 subjects with various LTF rs10865941 genotypes. The relationships among the LTF rs10865941 polymorphism, lactoferrin, Streptococcus mutans, and caries were investigated by using the ELISA and qRT-PCR, along with logistic regression analysis. RESULTS: The genotype distribution of the LTF gene were significantly different between the case and control groups (p = 0.018). The case group had lower C allele and greater T allele frequencies than the control group (p = 0.006). The LTF rs10865941 polymorphism was associated with caries in the codominant, dominant, and additive models (p < 0.05). MMP20 rs1784418, CA6 rs2274328, and TAS1R2 rs35874116 were not significantly different between the two groups (p > 0.05). A greater quantity of S. mutans. in the supragingival plaque was found in the case group (p = 0.03). There were significant differences between the two groups in both the codominant model and the dominant model (p < 0.05). CONCLUSIONS: The LTF rs10865941 polymorphism may be associated with caries susceptibility in the Zhuang population of China. The LTF rs10865941 T allele may be a potential risk factor for dental caries.

Molecular evolutionary analyses of tooth genes support sequential loss of enamel and teeth in baleen whales (Mysticeti).

The loss of teeth and evolution of baleen racks in Mysticeti was a profound transformation that permitted baleen whales to radiate and diversify into a previously underutilized ecological niche of bulk filter-feeding on zooplankton and other small prey. Ancestral state reconstructions suggest that postnatal teeth were lost in the common ancestor of crown Mysticeti. Genomic studies provide some support for this hypothesis and suggest that the genetic toolkit for enamel production was inactivated in the common ancestor of living baleen whales. However, molecular studies to date have not provided direct evidence for the complete loss of teeth, including their dentin component, on the stem mysticete branch. Given these results, several questions remain unanswered: (1) Were teeth lost in a single step or did enamel loss precede dentin loss? (2) Was enamel lost early or late on the stem mysticete branch? (3) If enamel and dentin/tooth loss were decoupled in the ancestry of baleen whales, did dentin loss occur on the stem mysticete branch or independently in different crown mysticete lineages? To address these outstanding questions, we compiled and analyzed complete protein-coding sequences for nine tooth-related genes from cetaceans with available genome data. Seven of these genes are associated with enamel formation (ACP4, AMBN, AMELX, AMTN, ENAM, KLK4, MMP20) whereas two other genes are either dentin-specific (DSPP) or tooth-specific (ODAPH) but not enamel-specific. Molecular evolutionary analyses indicate that all seven enamel-specific genes have inactivating mutations that are scattered across branches of the mysticete tree. Three of the enamel genes (ACP4, KLK4, MMP20) have inactivating mutations that are shared by all mysticetes. The two genes that are dentin-specific (DSPP) or tooth-specific (ODAPH) do not have any inactivating mutations that are shared by all mysticetes, but there are shared mutations in Balaenidae as well as in Plicogulae (Neobalaenidae + Balaenopteroidea). These shared mutations suggest that teeth were lost at most two times. Shared inactivating mutations and dN/dS analyses, in combination with cetacean divergence times, were used to estimate inactivation times of genes and by proxy enamel and tooth phenotypes at ancestral nodes. The results of these analyses are most compatible with a two-step model for the loss of teeth in the ancestry of living baleen whales: enamel was lost very early on the stem Mysticeti branch followed by the independent loss of dentin (and teeth) in the common ancestors of Balaenidae and Plicogulae, respectively. These results imply that some stem mysticetes, and even early crown mysticetes, may have had vestigial teeth comprised of dentin with no enamel. Our results also demonstrate that all odontocete species (in our study) with absent or degenerative enamel have inactivating mutations in one or more of their enamel genes.

The energetic basis for hydroxyapatite mineralization by amelogenin variants provides insights into the origin of amelogenesis imperfecta.

Small variations in the primary amino acid sequence of extracellular matrix proteins can have profound effects on the biomineralization of hard tissues. For example, a change in one amino acid within the amelogenin protein can lead to drastic changes in enamel phenotype, resulting in amelogenesis imperfecta, enamel that is defective and easily damaged. Despite the importance of these undesirable phenotypes, there is very little understanding of how single amino acid variation in amelogenins can lead to malformed enamel. Here, we aim to develop a thermodynamic understanding of how protein variants can affect steps of the biomineralization process. High-resolution, in situ atomic force microscopy (AFM) showed that altering one amino acid within the murine amelogenin sequence (natural variants T21 and P41T, and experimental variant P71T) resulted in an increase in the quantity of protein adsorbed onto hydroxyapatite (HAP) and the formation of multiple protein layers. Quantitative analysis of the equilibrium adsorbate amounts revealed that the protein variants had higher oligomer-oligomer binding energies. MMP20 enzyme degradation and HAP mineralization studies showed that the amino acid variants slowed the degradation of amelogenin by MMP20 and inhibited the growth and phase transformation of HAP. We propose that the protein variants cause malformed enamel because they bind excessively to HAP and disrupt the normal HAP growth and enzymatic degradation processes. The in situ methods applied to determine the energetics of molecular level processes are powerful tools toward understanding the mechanisms of biomineralization.

Spectrum of pathogenic variants and founder effects in amelogenesis imperfecta associated with MMP20.

Amelogenesis imperfecta (AI) describes a heterogeneous group of developmental enamel defects that typically have Mendelian inheritance. Exome sequencing of 10 families with recessive hypomaturation AI revealed four novel and one known variants in the matrix metallopeptidase 20 (MMP20) gene that were predicted to be pathogenic. MMP20 encodes a protease that cleaves the developing extracellular enamel matrix and is necessary for normal enamel crystal growth during amelogenesis. New homozygous missense changes were shared between four families of Pakistani heritage (c.625G>C; p.(Glu209Gln)) and two of Omani origin (c.710C>A; p.(Ser237Tyr)). In two families of UK origin and one from Costa Rica, affected individuals were homozygous for the previously reported c.954-2A>T; p.(Ile319Phefs*19) variant. For each of these variants, microsatellite haplotypes appeared to exclude a recent founder effect, but elements of haplotype were conserved, suggesting more distant founding ancestors. New compound heterozygous changes were identified in one family of the European heritage: c.809_811+12delinsCCAG; p.(?) and c.1122A>C; p.(Gln374His). This report further elucidates the mutation spectrum of MMP20 and the probable impact on protein function, confirms a consistent hypomaturation phenotype and shows that mutations in MMP20 are a common cause of autosomal recessive AI in some communities.

Effects of DSPP and MMP20 Silencing on Adhesion, Metastasis, Angiogenesis, and Epithelial-Mesenchymal Transition Proteins in Oral Squamous Cell Carcinoma Cells.

Recent reports highlight the potential tumorigenic role of Dentin Sialophosphoprotein (DSPP) and its cognate partner Matrix Metalloproteinase 20 (MMP-20) in Oral Squamous Cell Carcinomas (OSCCs). However, the function/mechanism of these roles is yet to be fully established. The present study aimed to investigate the effects of DSPP and MMP20 silencing on specific proteins involved in oral cancer cell adhesion, angiogenesis, metastasis, and epithelial-mesenchymal transition (EMT). Stable lines of DSPP/MMP20 silenced OSCC cell line (OSC2), previously established via lentiviral-mediated shRNA transduction, were analyzed for the effects of DSPP, MMP20, and combined DSPP-MMP20 silencing on MMP2, MMP9, integrins αvß3 and αvß6, VEGF, Kallikerin- 4,-5,-8,-10, E-cadherin, N-cadherin, Vimentin, met, src, snail, and Twist by Western blot. Results show a significant decrease (p < 0.05) in the expression of MMP2, MMP9, integrin αvß3, αvß6, VEGF, Kallikerins -4, -5, -8, -10, N-cadherin, vimentin met, src, snail and twist following DSPP and MMP20 silencing, individually and in combination. On the other hand, the expression of E-cadherin was found to be significantly increased (p < 0.05). These results suggest that the tumorigenic effect of DSPP and MMP20 on OSC2 cells is mediated via the upregulation of the genes involved in invasion, metastasis, angiogenesis, and epithelial-mesenchymal transition (EMT).

MMP20 Single-Nucleotide Polymorphisms Correlate with Susceptibility to Alcohol-Induced Osteonecrosis of the Femoral Head in Chinese Males.

BACKGROUND Alcohol-induced osteonecrosis of the femoral head (ONFH) is caused by the interaction of genetic and environmental factors. Genetic variations of matrix metalloproteinase (MMP) system are associated with ONFH development and progression. In this study, we aimed to evaluate the relationships between MMP20 gene polymorphisms and the risk of alcohol-induced ONFH in Chinese Han males. MATERIAL AND METHODS In this case-control study, genotypes of 14 selected SNPs in the MMP20 gene were assayed using MassARRAY in 299 male cases with alcohol-induced ONFH and in 197 healthy males. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to assess the influence of gene polymorphism on occurrence of alcohol-induced ONFH by allelic model analysis, genotype model analysis and haplotype analysis. RESULTS After allelic model analysis, the minimum alleles of rs10895322, rs1784424, rs3781788, and rs1573954 correlated with an increased risk of alcohol-induced ONFH (P<0.05). Genetic model analysis revealed significant associations of 9 SNPs with alcohol-induced ONFH occurrence even after adjustment for age (P<0.05): 2 protective SNPs (rs1711423 and rs1784418) and 7 high-risk SNPs (rs10895322, rs1784424, rs3781788, rs7126560, rs1573954, rs1711399, and rs2292730). Moreover, 8 SNPs showed a statistically significant association with different clinical phenotypes (P<0.05). Beyond that, haplotype "CGGTTCCA" in MMP20 was discovered to correlate with a 1.63-fold increased risk of alcohol-induced ONFH (OR: 1.63, 95% CI: 1.15-2.30, P=0.0058). CONCLUSIONS Our data sheds new light on the associations of MMP20 gene polymorphisms with alcohol-induced ONFH predisposition in Chinese Han males.

Analysis of Polymorphisms in Genes Differentially Expressed in the Enamel of Mice with Different Genetic Susceptibilities to Dental Fluorosis.

Genes expressed during amelogenesis are candidates to increase the risk of dental fluorosis (DF). Thus, this study aimed to evaluate the association between polymorphisms in enamel development genes and susceptibility to DF in mice. Mice of both sexes, representing strains 129P3/J (n = 20; resistant to DF) and A/J (n = 20; susceptible to DF), were divided into 2 groups. Each strain received a diet with a low concentration of fluoride (F) and drinking water containing 0 or 50 mg/L of F for 6 weeks. Clinical evaluation and analysis of Vickers enamel microhardness of the incisors were performed. Livers were collected for genomic DNA extraction. Seventeen genetic polymorphisms in Amelx, Ambn, Ambn, Col14a1, Col1a1, Col5a2, Enam, Fam20a, Fam83h, Foxo1, Klk4, Mmp20, Serpinf1, Serpinh1, Smad3, Tuft1, and Wdr72 were genotyped by real-time PCR using Taqman chemistry. Overrepresentation of alleles and genotypes in DF was evaluated using the χ2 test with an alpha of 5%. The clinical aspects of the enamel and the surface enamel microhardness confirmed the DF condition. In the polymorphisms rs29569969, rs13482592, and rs13480057 in Ambn, Col14a1, and Mmp20, respectively, genotype and allele distributions were statistically significantly different between A/J and 129P3/J strains (p < 0.05). In conclusion, polymorphisms in Ambn, Col14a1, and Mmp20 are associated with the susceptibility to DF.

DSPP-MMP20 gene silencing downregulates cancer stem cell markers in human oral cancer cells.

BACKGROUND: Recent findings indicate that dentin sialophosphoprotein (DSPP) and matrix metalloproteinase (MMP) 20 interact in oral squamous cell carcinoma (OSCC). The objective of this study was to determine the effects of DSPP/MMP20 gene silencing on oral cancer stem cell (OCSC) markers. METHODS: The expression of well-established OCSC markers: ABCG2; ALDH1; CD133; CD44; BMI1; LGR4, and Podoplanin in DSPP/MMP20-silenced OSCC cell line, OSC2, and controls were assayed by western blot (WB), and flow cytometry techniques. The sensitivity of OSC2 cells to cisplatin following DSPP/MMP20 silencing was also determined. RESULTS: DSPP/MMP20 silencing resulted in downregulation of OCSC markers, more profoundly ABCG2 (84%) and CD44 (81%), following double silencing. Furthermore, while treatment of parent (pre-silenced) OSC2 cells with cisplatin resulted in upregulation of OCSC markers, DSPP/MMP20-silenced OSC2 cells similarly treated resulted in profound downregulation of OCSC markers (72 to 94% at 50 µM of cisplatin), and a marked reduction in the proportion of ABCG2 and ALDH1 positive cells (~ 1%). CONCLUSIONS: We conclude that the downregulation of OCSC markers may signal a reduction in OCSC population following MMP20/DSPP silencing in OSCC cells, while also increasing their sensitivity to cisplatin. Thus, our findings suggest a potential role for DSPP and MMP20 in sustaining OCSC population in OSCCs, possibly, through mechanism(s) that alter OCSC sensitivity to treatment with chemotherapeutic agents such as cisplatin.

Effects of Er:YAG and Diode Laser Irradiation on Dental Pulp Cells and Tissues.

Vital pulp therapy (VPT) is to preserve the nerve and maintain healthy dental pulp tissue. Laser irradiation (LI) is beneficial for VPT. Understanding how LI affects dental pulp cells and tissues is necessary to elucidate the mechanism of reparative dentin and dentin regeneration. Here, we show how Er:YAG-LI and diode-LI modulated cell proliferation, apoptosis, gene expression, protease activation, and mineralization induction in dental pulp cells and tissues using cell culture, immunohistochemical, genetic, and protein analysis techniques. Both LIs promoted proliferation in porcine dental pulp-derived cell lines (PPU-7), although the cell growth rate between the LIs was different. In addition to proliferation, both LIs also caused apoptosis; however, the apoptotic index for Er:YAG-LI was higher than that for diode-LI. The mRNA level of odontoblastic gene markers-two dentin sialophosphoprotein splicing variants and matrix metalloprotease (MMP)20 were enhanced by diode-LI, whereas MMP2 was increased by Er:YAG-LI. Both LIs enhanced alkaline phosphatase activity, suggesting that they may help induce PPU-7 differentiation into odontoblast-like cells. In terms of mineralization induction, the LIs were not significantly different, although their cell reactivity was likely different. Both LIs activated four MMPs in porcine dental pulp tissues. We helped elucidate how reparative dentin is formed during laser treatments.

MMP20 rs1784418 Protects Certain Populations against Caries.

This work aimed to further evaluate the association of MMP20 rs1784418 C>T and dental caries experience with the hypothesis that MMP20 rs1784418 C>T is a risk factor for dental caries. 184 children 4-7 years of age had their caries experience determined and buccal cheek swabs collected for DNA extraction to test for association with the MMP20 rs1784418 C>T using standard statistical approaches. A meta-analytic approach was also implemented to compile previous discrepant reports of the same association. We found an association between MMP20 rs1784418 C>T and dental caries experience in primary dentition (p = 0.01). The meta-analysis showed that this association appears to favor individuals born in Brazil and not Turkey. MMP20 rs1784418 C>T appears to protect against dental caries, but its effects are likely to be more marked in certain populations.

MMP20 and ARMS2/HTRA1 Are Associated with Neovascular Lesion Size in Age-Related Macular Degeneration.

PURPOSE: Age-related macular degeneration (AMD) is the leading cause of severe visual impairment. Despite treatment, a central scotoma often remains. The size of the scotoma depends on the lesion size of the choroidal neovascular membrane and significantly affects the patient's quality of life, and the lesion size of neovascularization also affects response to treatments. The aim of this study was to identify genes associated with the neovascular lesion size in neovascular AMD. DESIGN: A genome-wide association study (GWAS). PARTICIPANTS: We included 1146 Japanese patients with neovascular AMD. METHODS: We performed a 2-stage GWAS for the lesion size of AMD as a quantitative trait among 1146 (first stage: 727, second stage: 419) Japanese patients with neovascular AMD. Lesion size was determined by the greatest linear dimension measured with fluorescein angiography examination before treatment. We examined the association between the genotypic distribution of each single nucleotide polymorphism (SNP) and the trait using an additive model adjusted for age and sex. To evaluate the associations between AMD development and SNPs associated with lesion size, we also performed a case-control study by using the genotype data from these 1146 Japanese patients as case subjects and the fixed dataset from the Nagahama Study as control subjects. MAIN OUTCOME MEASURES: Genes associated with the lesion size in neovascular AMD. RESULTS: In the discovery stage, rs10895322 in MMP20 showed a genome-wide significant P value of 6.95×10(-8), and rs2284665 in ARMS2/HTRA1 showed a P value of 1.55×10(-7). The associations of these 2 SNPs were successfully replicated in the replication stage, and a meta-analysis of both stages showed genome-wide significant P values (2.80×10(-9) and 4.41×10(-9), respectively). In a case-control study using 3248 Japanese subjects as controls, we could not find contribution of MMP20 rs10895322 for AMD development. Although MMP20 has been thought to be expressed only in dental tissues, we confirmed MMP20 expression in the human retina and retinal pigment epithelium/choroid with polymerase chain reaction. CONCLUSIONS: The growth of choroidal neovascularization in AMD would be affected by 2 genes: MMP20, a newly confirmed gene expressed in the retina, and ARMS2/HTRA1, a well-known susceptibility gene for AMD.

Genetic comparisons yield insight into the evolution of enamel thickness during human evolution.

Enamel thickness varies substantially among extant hominoids and is a key trait with significance for interpreting dietary adaptation, life history trajectory, and phylogenetic relationships. There is a strong link in humans between enamel formation and mutations in the exons of the four genes that code for the enamel matrix proteins and the associated protease. The evolution of thick enamel in humans may have included changes in the regulation of these genes during tooth development. The cis-regulatory region in the 5' flank (upstream non-coding region) of MMP20, which codes for enamelysin, the predominant protease active during enamel secretion, has previously been shown to be under strong positive selection in the lineages leading to both humans and chimpanzees. Here we examine evidence for positive selection in the 5' flank and 3' flank of AMELX, AMBN, ENAM, and MMP20. We contrast the human sequence changes with other hominoids (chimpanzees, gorillas, orangutans, gibbons) and rhesus macaques (outgroup), a sample comprising a range of enamel thickness. We find no evidence for positive selection in the protein-coding regions of any of these genes. In contrast, we find strong evidence for positive selection in the 5' flank region of MMP20 and ENAM along the lineage leading to humans, and in both the 5' flank and 3' flank regions of MMP20 along the lineage leading to chimpanzees. We also identify putative transcription factor binding sites overlapping some of the species-specific nucleotide sites and we refine which sections of the up- and downstream putative regulatory regions are most likely to harbor important changes. These non-coding changes and their potential for differential regulation by transcription factors known to regulate tooth development may offer insight into the mechanisms that allow for rapid evolutionary changes in enamel thickness across closely-related species, and contribute to our understanding of the enamel phenotype in hominoids.

Transforming growth factor-ß1 regulates expression of the matrix metalloproteinase 20 (Mmp20) gene through a mechanism involving the transcription factor, myocyte enhancer factor-2C, in ameloblast lineage cells.

Matrix metalloproteinase-20 (Mmp20) plays an essential role in amelogenesis during tooth development and is regulated by transforming growth factor-ß1 (TGF-ß1) in mouse ameloblast lineage cells (ALCs). The objective of this study was to explore the role of myocyte enhancer factor-2C (MEF2C), a key transcription factor in craniofacial development, in TGF-ß1-induced Mmp20 gene expression. We investigated Mmp20 expression in ALCs over-expressing MEF2C and in ALCs with MEF2C knocked down. We also analyzed activity of the Mmp20 promoter using a transient reporter gene-expression assay in cultured ALCs. Putative transcription factor-binding sites for MEF2C and TGF-ß1 on the Mmp20 promoter were analyzed with bioinformatics tools and examined using an electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). The expression of Mmp20 was induced, in a dose-dependent manner, by MEF2C over-expression, and TGF-ß1-induced Mmp20 expression was blocked by MEF2C knockdown in ALCs. There was a TGF-ß1/MEF2C-responsive region, including a putative MEF2-binding site, between base pairs -356 and -73 of the Mmp20 promoter. Mutation of the putative MEF2-binding site significantly reduced Mmp20 promoter activity upon activation with MEF2C or TGF-ß1. In conclusion, TGF-ß1-induced Mmp20 expression in ALCs was regulated through the MEF2-binding site on the Mmp20 promoter and thus mediated by the MEF2C signaling pathway.

Effect of kallikrein 4 loss on enamel mineralization: comparison with mice lacking matrix metalloproteinase 20.

Enamel formation depends on a triad of tissue-specific matrix proteins (amelogenin, ameloblastin, and enamelin) to help initiate and stabilize progressively elongating, thin mineral ribbons of hydroxyapatite formed during an appositional growth phase. Subsequently, these proteins are eradicated to facilitate lateral expansion of the hydroxyapatite crystallites. The purpose of this study was to investigate changes in enamel mineralization occurring in mice unable to produce kallikrein 4 (Klk4), a proteinase associated with terminal extracellular degradation of matrix proteins during the maturation stage. Mice lacking functional matrix metalloproteinase 20 (Mmp20), a proteinase associated with early cleavage of matrix proteins during the secretory stage, were also analyzed as a frame of reference. The results indicated that mice lacking Klk4 produce enamel that is normal in thickness and overall organization in terms of layers and rod/inter-rod structure, but there is a developmental defect in enamel rods where they first form near the dentinoenamel junction. Mineralization is normal up to early maturation after which the enamel both retains and gains additional proteins and is unable to mature beyond 85% mineral by weight. The outmost enamel is hard, but inner regions are soft and contain much more protein than normal. The rate of mineral acquisition overall is lower by 25%. Mice lacking functional Mmp20 produce enamel that is thin and structurally abnormal. Relatively high amounts of protein remain throughout maturation, but the enamel is able to change from 67 to 75% mineral by weight during maturation. These findings reaffirm the importance of secreted proteinases to enamel mineral acquisition.

Tributyltin alters osteocalcin, matrix metalloproteinase 20 and dentin sialophosphoprotein gene expression in mineralizing mouse embryonic tooth in vitro.

We showed in a previous in vitro study that tributyltin (TBT) arrests dentin mineralization and enamel formation in developing mouse tooth. The present aim was to investigate the effect of TBT on the expression of genes associated with mineralization of dental hard tissues. Embryonic day 18 mouse mandibular first molars were cultured for 3, 5 or 7 days and exposed to 1.0 µM TBT and studied by real-time quantitative polymerase chain reaction (RT-QPCR) for the expressions of osteocalcin (Ocn), alkaline phosphatase (Alpl), dentin matrix protein 1 (Dmp1), dentin sialophosphoprotein (Dspp) and matrix metalloproteinase 20 (Mmp-20).Ocn, Mmp-20 and Dspp, whose expressions showed changes in RT- QPCR, were further analyzed by in situ hybridization of tissue sections. In situ hybridization showed that TBT decreased Ocn expression in odontoblasts but increased the expression in the epithelial tooth compartment. In QPCR assays, the net effect in the whole tooth was increased expression. TBT also reduced Mmp-20 expression in ameloblasts and odontoblasts. Dspp expression varied but both QPCR assays and in situ hybridization showed a decreasing trend. TBT exposure had no clear effect on Alpl and Dmp1 expressions. Increased Ocn expression by epithelial enamel organ may inhibit dentin mineralization and enamel formation. Decreased Ocn, Mmp-20 and Dspp expressions in odontoblasts may indicate delayed cell differentiation, or TBT may specifically decrease the expression of genes involved in dentin mineralization. While decreased Mmp-20 expression by TBT in ameloblasts may impair enamel mineralization, the coincident reduction in Mmp-20 and Dspp expressions in odontoblasts may potentiate the delay of dentin mineralization.

Sequential use of transcriptional profiling, expression quantitative trait mapping, and gene association implicates MMP20 in human kidney aging.

Kidneys age at different rates, such that some people show little or no effects of aging whereas others show rapid functional decline. We sequentially used transcriptional profiling and expression quantitative trait loci (eQTL) mapping to narrow down which genes to test for association with kidney aging. We first performed whole-genome transcriptional profiling to find 630 genes that change expression with age in the kidney. Using two methods to detect eQTLs, we found 101 of these age-regulated genes contain expression-associated SNPs. We tested the eQTLs for association with kidney aging, measured by glomerular filtration rate (GFR) using combined data from the Baltimore Longitudinal Study of Aging (BLSA) and the InCHIANTI study. We found a SNP association (rs1711437 in MMP20) with kidney aging (uncorrected p = 3.6 x 10(-5), empirical p = 0.01) that explains 1%-2% of the variance in GFR among individuals. The results of this sequential analysis may provide the first evidence for a gene association with kidney aging in humans.

A multidisciplinary approach for the diagnosis of hypocalcified amelogenesis imperfecta in two Chilean families.

OBJECTIVE: The purpose of this study was to conduct a multidisciplinary analysis of a specific type of tooth enamel disturbance (amelogenesis imperfecta) affecting two Chilean families to obtain a precise diagnosis and to investigate possible underlying mutations. MATERIALS AND METHODS: Two non-related families affected with amelogenesis imperfecta were evaluated with clinical, radiographic and histopathological methods. Furthermore, pedigrees of both families were constructed and the presence of eight mutations in the enamelin gene (ENAM) and three mutations in the enamelysin gene (MMP-20) were investigated by PCR and direct sequencing. RESULTS: In the two affected patients, the dental malformation presented as soft and easily disintegrated enamel and exposed dark dentin. Neither of the affected individuals presented with a dental and skeletal open bite. Histologically, a high level of an organic matrix with prismatic organization was found. Genetic analysis indicated that the condition is autosomal recessive in one family and either autosomal recessive or due to a new mutation in the other family. Molecular mutational analysis revealed that none of the eight mutations previously described in the ENAM gene or the three mutations in the MMP-20 gene were present in the probands. CONCLUSION: A multidisciplinary analysis allowed for a diagnosis of hypocalcified amelogenesis imperfecta, Witkop type III, which was unrelated to previously described mutations in the ENAM or MMP-20 genes.

Pseudogenization of the tooth gene enamelysin (MMP20) in the common ancestor of extant baleen whales.

Whales in the suborder Mysticeti are filter feeders that use baleen to sift zooplankton and small fish from ocean waters. Adult mysticetes lack teeth, although tooth buds are present in foetal stages. Cladistic analyses suggest that functional teeth were lost in the common ancestor of crown-group Mysticeti. DNA sequences for the tooth-specific genes, ameloblastin (AMBN), enamelin (ENAM) and amelogenin (AMEL), have frameshift mutations and/or stop codons in this taxon, but none of these molecular cavities are shared by all extant mysticetes. Here, we provide the first evidence for pseudogenization of a tooth gene, enamelysin (MMP20), in the common ancestor of living baleen whales. Specifically, pseudogenization resulted from the insertion of a CHR-2 SINE retroposon in exon 2 of MMP20. Genomic and palaeontological data now provide congruent support for the loss of enamel-capped teeth on the common ancestral branch of crown-group mysticetes. The new data for MMP20 also document a polymorphic stop codon in exon 2 of the pygmy sperm whale (Kogia breviceps), which has enamel-less teeth. These results, in conjunction with the evidence for pseudogenization of MMP20 in Hoffmann's two-toed sloth (Choloepus hoffmanni), another enamel-less species, support the hypothesis that the only unique, non-overlapping function of the MMP20 gene is in enamel formation.

Molecular evolution of matrix metalloproteinase 20.

Dental enamel is a hypermineralized tissue, containing only trace amounts of organic components. During enamel formation, matrix metalloproteinase 20 (MMP20) processes proteins comprising enamel matrix and facilitates hypermineralization. In the human genome, 24 distinct MMP genes have been identified. Among these genes, MMP20 is clustered with eight other genes, including MMP13, and all these clustered genes show phylogenetically close relationships. In this study, we investigated MMP20 and closely related MMP genes in various tetrapods and in a teleost fish, fugu. In the genome of the chicken, a toothless tetrapod, we identified degraded exons of MMP20, which supports the previous proposition that MMP20 is important specifically for enamel formation. Nevertheless, for unknown reasons, we failed to identify MMP20 in the platypus genome. In the opossum, lizard, and frog genomes, MMP20 was found clustered with MMP13. Furthermore, in the fugu genome, we identified an MMP20-like gene located adjacent to MMP13, suggesting that MMP20 arose before the divergence of ray-finned fish and lobe-finned fish. The teleost tooth surface is covered with enameloid, a hypermineralized tissue different from enamel. Thus, we hypothesize that MMP20 could have been used in an ancient hypermineralized tissue, which evolved into enameloid in teleosts and into enamel in tetrapods.

Target gene analyses of 39 amelogenesis imperfecta kindreds.

Previously, mutational analyses identified six disease-causing mutations in 24 amelogenesis imperfecta (AI) kindreds. We have since expanded the number of AI kindreds to 39, and performed mutation analyses covering the coding exons and adjoining intron sequences for the six proven AI candidate genes [amelogenin (AMELX), enamelin (ENAM), family with sequence similarity 83, member H (FAM83H), WD repeat containing domain 72 (WDR72), enamelysin (MMP20), and kallikrein-related peptidase 4 (KLK4)] and for ameloblastin (AMBN) (a suspected candidate gene). All four of the X-linked AI families (100%) had disease-causing mutations in AMELX, suggesting that AMELX is the only gene involved in the aetiology of X-linked AI. Eighteen families showed an autosomal-dominant pattern of inheritance. Disease-causing mutations were identified in 12 (67%): eight in FAM83H, and four in ENAM. No FAM83H coding-region or splice-junction mutations were identified in three probands with autosomal-dominant hypocalcification AI (ADHCAI), suggesting that a second gene may contribute to the aetiology of ADHCAI. Six families showed an autosomal-recessive pattern of inheritance, and disease-causing mutations were identified in three (50%): two in MMP20, and one in WDR72. No disease-causing mutations were found in 11 families with only one affected member. We conclude that mutation analyses of the current candidate genes for AI have about a 50% chance of identifying the disease-causing mutation in a given kindred.