Clinical and radiographic examinations of the probands and their available family members were performed, and blood samples were collected with the understanding and written consent of each participant according to the Declaration of Helsinki. Affected individuals were healthy, except hypomaturation enamel defects. The study protocol was independently reviewed and approved by the Institution Review Board at the Seoul National University Dental Hospital, the University of Istanbul and the University of Michigan.

Genomic DNA was isolated from peripheral whole blood. The purity and concentration of the DNA were quantified by spectrophotometry measurement and the OD₂₆₀/OD₂₈₀ ratio obtained. Whole-exome sequencing was performed with the DNA sample of the probands using Illumina HiSeq 2000 platform. The NimbleGen (Roche Diagnostics, Indianapolis, IL, USA) exome capture reagent was used for exome capturing.

The affected individuals in family 1 (IV:3 and IV:4) were genotyped with the Affymetrix Genome-Wide Human SNP array 6.0 (DNALINK INC., Seoul, Korea). The annotated SNP files were analyzed with HomozygosityMapper (http://www.homozygositymapper.org/) (Seelow et al., 2009) to identify the shared regions of homozygosity in the affected individuals.

The sequence variations in the MMP20 gene and segregation within each family was confirmed by Sanger sequencing with primers and conditions described previously (Kim et al., 2005). PCR amplifications were done with the HiPi DNA polymerase premix (Elpis Biotech, Daejeon, Korea), and DNA sequencing was performed at a DNA sequencing center (Macrogen, Seoul, Korea).

Human MMP20 cDNA, previously cloned into the pcDNA3.1 vector, was used to introduce the identified mutations using PCR mutagenesis (sense: 5′-TACCGTTGCTGCTCAAGAATTTGGCCATGC, antisense: 5′-GCATGGCCAAATTCTTGAGCAGCAACGGTA for the p.His226Gln and sense: 5′-GAATATCTAAATACATACCTTCCATGAGTT, antisense: 5′-AACTCATGGAAGGTATGTATTTAGATATTC for the p.Thr130Ile) (Lee et al., 2010). Sequences of normal and mutant MMP20 pcDNA3.1 vectors were confirmed by direct plasmid sequencing.

HEK293T cells were grown and maintained in DMEM supplemented with 10% FBS and antibiotics in a 5% CO₂ atmosphere at 37°C. Cells at ~2 × 10⁵ quantity were seeded in each well of the 6-well culture dish. Each plasmid construct at 2 ug quantity was transiently transfected into HEK293T cells with Genjet in vitro DNA transfection reagent (SigmaGen Laboratories, Ijamsville, MD, USA). The culture medium of each well was harvested after 30 h of incubation and concentrated using Amicon ultra-4 centrifugal filter units (Millipore, Bedford, MA, USA).

Four ml of conditioned medium from the culture was collected and concentrated to 50 ul. The concentrated media of 20 ul was mixed with 4 ul of 5x non-reducing buffer, then loaded onto the 11% SDS-polyacrylamide gel with β-casein (Sigma-Aldrich, St. Louis, MO, USA) as a substrate. The zymogram was developed, stained with Coomassie brilliant blue R-250 staining solution (Bio-rad, Hercules, CA, USA) for 20 min, and visualized after incubation in a destaining solution (10% MeOH, 10% acetic acid) for 3 h.

Concentrated media and cell lysates were run on the 11% SDS-polyacrylamide gel and subjected to Western blotting. Specifically, 50 ug cell lysate from each sample and 10 ul of concentrated media were used. After gel transfer to the PVDF membrane and blocking, MMP20 was detected by incubating the membrane with primary antibody overnight at 4°C and with secondary antibody for 2 h at room temperature. The primary antibodies used were a rabbit polyclonal anti-MMP20 antibody (ab39038, abcam plc., Cambridge, UK) and a mouse monoclonal anti-ACTB antibody (A2228, Sigma-Aldrich, St. Louis, MO, USA); both of which were diluted in 1:10,000.

The proband of Family 1 (IV:4) was an 11-year-old girl from a consanguineous marriage of first cousins (Figure 1A). Her prenatal and perinatal history was uneventful and her parents reported no other medical problem. Her teeth exhibited generalized brown discoloration with exogenous black pigmentation mainly on occlusal surface of the posterior teeth (Figures 1B–D). Maxillary left central incisor was lost due to trauma. The radiopacity of enamel did not contrast well with dentin in the panoramic radiograph, consistent with hypomineralization (Figure 1E). Her 24-year-old brother (IV:3) was also affected and almost all of his teeth have been reconstructed with full-coverage prosthodontics. His remaining natural teeth exhibited dark brown discoloration with exogenous pigmentation (Figure S1).

The proband of the family 2 (III:1) was a 10-year-old girl from a non-consanguineous family (Figure 2A). Her past medical history was unremarkable. Her anterior permanent teeth were not severely discolored, but slightly yellow and less transparent than normal teeth (Figures 2B,C). Her right second premolar was congenitally absent based on the panoramic radiograph (Figure 2D).

Sequencing reads were aligned to the UCSC human reference genome (hg19) with Burrow-Wheeler Aligner, and the sequence variations were annotated by referencing dbSNP build 138, which preceded variant calling with SAMtools and GATK (Table S1). Annotated variants were filtered with the criteria of minor allele frequency of 0.01.

Autozygosity mapping of the family 1 revealed 3 shared regions of loss of heterozygosity: chr4:65,904,881–82,427,846, chr11:83,358,629–113,318,007, and chr21:11,039,570–17,728,224 (Figure S2). The exome data in the shared regions of the proband in family 1 revealed a homozygous variant in exon 5 of the MMP20 gene (NM_004771.3: c.678T>A). This transversion of thymine to adenine changed histidine to glutamine at amino acid position 226 (p.His226Gln). There was no other variation in the known AI-causing genes and the MMP20 mutation (c.678T>A, p.His226Gln) was previously reported as an AI-causing mutation (Ozdemir et al., 2005; Wright et al., 2011).

Whole exome sequencing of the Family 2 proband revealed compound heterozygous MMP20 mutations (c.389C>T and c.540T>A). There was no other variation in the known AI-causing genes. A cytosine to thymine transition in exon 3 changed threonine to isoleucine at amino acid position 130 (p.Thr130Ile). This variation was listed in the Exome Aggregation Consortium (ExAC) database (rs61730849) with an allele frequency of 0.00165 (200/121176). But the frequency was relatively high (0.0294) in a certain subset of small population (ss86247256, AGI_ASP_population; Coriell Apparently Healthy Collection). In addition, it was previously reported as a disease-causing mutation (Gasse et al., 2013). The other variation, a transversion of thymine to adenine in exon 4, would introduce a premature stop codon (p.Tyr180^*) and the mutant transcript would be degraded by the nonsense-mediated decay system. This variant was not listed in any database.

Segregation within the families by Sanger sequencing confirmed that the nonsense mutation (c.540T>A, p.Tyr180^*) was transmitted paternally and the missense mutation (c.389C>T, p.Thr130Ile) was transmitted maternally to the proband (Figure 3A, Figure S3). These amino acids at the mutation sites (Thr130, Tyr180, and His226) are strictly conserved among eutherian mammal orthologs (Figure 3B).

Western blotting and zymography determining the function of the MMP20 mutants demonstrated that the p.Thr130Ile mutant protein was secreted at a reduced amount and had proteolytic activity. Western blot of cell lysate revealed that the p.His226Gln mutant protein was retained in the cell and likely not able to be secreted (Figures 3C,D).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.