A Novel De Novo SP6 Mutation Causes Severe Hypoplastic Amelogenesis Imperfecta

Abstract

Amelogenesis imperfecta (AI) is a heterogeneous group of rare genetic disorders affecting tooth enamel formation. Here we report an identification of a novel de novo missense mutation [c.817_818delinsAT, p.(Ala273Met)] in the SP6 gene, causing non-syndromic autosomal dominant AI. This is the second paper on amelogenesis imperfecta caused by SP6 mutation. Interestingly the identified mutation in this study is a 2-bp variant at the same nucleotide positions as the first report, but with AT instead of AA insertion. Clinical phenotype was much more severe compared to the previous report, and western blot showed an extremely decreased level of mutant protein compared to the wild-type, even though the mRNA level was similar.

1. Introduction

Amelogenesis imperfecta (AI) is a collection of hereditary enamel defects that are genetically heterogeneous and phenotypically diverse [1]. The prevalence varies from 1:700 to 1:14,000, according to the location and study population [2]. Furthermore, AI can occur as an isolated condition or as part of a syndromic phenotype [3]. Clinically, AI can be categorized as a hypoplastic, hypocalcification or hypomaturation form, depending on the quantity and quality of the affected enamel. However, often the clinical phenotype is not clear enough to derive the correct diagnosis of AI, primarily because of the inadequate number of samples for analyses or because the destruction of the tooth structure, due to attrition, abrasion, or dental caries, has obscured the clinical presentation of the enamel defects [4,5]. Therefore, the term “hypomineralization” was proposed to indicate hypocalcification and hypomaturation, together [6].

Mutations in genetic factors such as DLX3 or AMELX expressed during early amelogenesis can have a profound impact on the entire enamel forming process, resulting in AI with both hypoplastic and hypomaturation features [7,8,9]. Furthermore, the clinical phenotype of AI may be altered by differences in genetic background and influences from environmental factors such as systemic and/or nutritional disturbances [10].

To date, more than 20 genes have been identified to cause AI [11]. Genes encoding the structural proteins (AMELX, ENAM and AMBN) and proteolytic enzymes (MMP20 and KLK4) in the developing enamel matrix have been screened initially, and mutations have been identified [12,13,14,15,16]. With the remarkable advances in modern genetic techniques, such as whole exome or genome sequencing, many genes with little-known or unknown functions in amelogenesis have been successfully identified [4,17].

Recently, the involvement of a novel gene, SP6 (aka, Epiprofin or EPFN), was identified in a Caucasian family to cause autosomal dominant hypoplastic AI [18]. In this study, we report the identification of the second SP6 mutation in a Korean AI family and present a unique clinical phenotype.

2. Materials and Methods

2.1. Study Subjects and Informed Consent

The institutional review board of the Seoul National University Dental Hospital reviewed and approved the study protocol (CRI05003G), and informed consent was obtained from each individual or a guardian. Clinical and radiographic examination were performed

2.2. Clinical Examination and Sample Collection

The proband, mother and father were examined for complete oral and systemic evaluation. The older brother of the proband was reportedly healthy, without any tooth symptoms, and could not attend the examination; therefore, a saliva DNA collection kit (Oragene OG-250, DNAgenotek, Ottawa, Ontario, Canada) was given to him. Peripheral whole blood was collected from the other family members.

2.3. DNA Isolation and Whole Exome Sequencing

Genomic DNA was isolated from 2 ml of peripheral whole blood or saliva using the NucleoSpin genomic DNA purification kit (Macherey-Nagel GmbH & Co., Düren, Germany). The purified DNA was measured for quality and quantity by spectrophotometry measured by the OD₂₆₀/OD₂₈₀ ratio. To further check the integrity of the isolated DNA, agarose gel electrophoresis was performed. Whole exome sequencing was performed with the Agilent SureSelect XT Human All Exon V5 Target Enrichment System, and paired-end sequencing reads were obtained with the Illumina HiSeq 2500 (Theragen Etex Bio Institute, Suwon-si, Korea).

2.4. Bioinformatics

A series of bioinformatic analysis programs were applied to the obtained paired-end sequencing reads. The sequencing reads were trimmed to remove the adapter sequences using Cutadapt and aligned to the reference human genome assembly hg38 with the Burrows-Wheeler Aligner. Samtools, Genome Analysis Tool Kit, and Annovar were used to obtain a list of sequence variants. Sequence variants were annotated with dbSNP build 147.

2.5. Sanger Sequencing

The identified mutation in the proband was confirmed by Sanger sequencing with the following primers (676 bp, sense: 5′-GGGCTAAGGCCTTGGAAGTA-3′, antisense: 5′-AAATACGCACCTTCCCCTCT-3′). Sanger sequencing was performed for all four participating family members.

2.6. Cloning of the Wild-Type and Mutant SP6 Expression Vectors

The wild-type SP6 coding sequence with flag tagging was cloned from the genomic DNA with the following primers (sense: 5′-AAGCTTGATCCCGGCAATGCTAACCGC-3′, antisense: 5′-CAGCGTGGCTCCCTCCAACTCAGCCGACTACAAAGACGATGACGACAAGCTCGAG-3′), and the mutation was introduced by conventional PCR mutagenesis (sense: 5′-ACGTCGCACCTGAAGATGCACCTGCGCTGG-3′, antisense: 5′-CCAGCGCAGGTGCATCTTCAGGTGCGACGT-3′). Wild-type and mutant sequences were subcloned into the pcDNA3.1 vector with HindIII and XhoI restriction endonucleases.

2.7. Western Blot and RT-PCR of the Wild-Type and Mutant SP6

COS-7 cells in 35 mm culture dishes were transfected with the wild-type and mutant SP6 vectors. After 30 hours post-transfection, the cells were harvested with RIPA buffer with protease inhibitors. Primary antibodies for flag (Sigma-Aldrich, MO, USA) and GAPDH (abm, Richmond, BC, Canada) were used at a titer of 1:10,000 and incubated at 4 °C overnight. The secondary antibody of goat anti-mouse (Thermo, Waltham, MA, USA), conjugated with HRP, was used at a titer of 1:10000. Total RNA was isolated with the RNeasy mini Kit (Qiagen, Germantown, MD, USA), and cDNA was synthesized. RT-PCR was performed using the SP6 primers (530 bp, sense: 5′-GGTAACCTGCGAGGACCTG-3′, antisense: 5′-GCTTCTTCTTGCCCCCATC-3′) and the GAPDH primers (309 bp, sense: 5′-CCAAGGTCATCCATGACAAC-3′, antisense: 5′-GCTTCACTACCTTCTTGATG-3′).

3. Results

The proband was a nine-year-old boy with hypoplastic AI who was referred from a local dental clinic. He was the second child from a non-consanguineous marriage and uneventful pregnancy and delivery (Figure 1a). His height and weight were slightly small compared to the same age group. His height (130.2 cm) was 9.5 percentile and his weight (26.5 kg) was 7.3 percentile, according to the Korean reference growth chart. He received surgery to correct bilateral blepharoptosis when he was six years old. Otherwise, his past medical history was unremarkable, and there was no other individual having similar dental features on the paternal or maternal sides of the family. To manage the rapidly wearing tooth structure, the first permanent molars and deciduous molars were treated with stainless steel crowns. The hypoplastic enamel, resulting in small teeth with wide interdental spacing, can be observed in the anterior region of the mouth (Figure 1b). Panoramic radiograph examination revealed severe hypoplastic enamel in all developing teeth and uniquely peculiar rocket-shaped developing roots of the canines and premolars (Figure 2a). After full eruption, these teeth showed a blunt root apex with enlarged pulp chambers. The first permanent molars exhibited taurodontism and a vertically extended pulp chamber resulting in short roots. Later, the taurodontism was also clearly seen in the second molars (Figure 2b).

Whole exome sequencing and trio analysis identified a likely pathogenic variant (a de novo SP6 mutation) without any of the other mutations in the known AI-causing genes (

Table 1. Statistics for whole exome sequencing.

Sample	Total Reads	Mapping Rate (%)	Median Target Coverage	Coverage of Target Region (%)	Fraction of Target Covered with at Least
					20X	10X
I:1	69,365,294	99.6	95	96.5	93.5	95.5
I:2	68,921,817	99.6	87	96.3	93.0	95.3
II:2	59,890,807	99.7	82	96.5	92.8	95.4

). The identified mutation was a 2-bp insertion and deletion mutation (NM_199262.3: c.817_818delinsAT), which would be predicted to change the highly conserved amino acid alanine to methionine at codon position 273 [NP_954871.1: p.(Ala273Met)] (Figure 3a). This mutation was not listed in the Exome Aggregation Consortium (ExAC) and gnomAD databases. In silico prediction programs, PolyPhen-2 and SIFT predicted probably damaging with a score of one and deleterious with a score of zero, respectively. Furthermore, the protein expression study with western blot revealed that the expression of the mutant protein was reduced compared to the wild type, even though their mRNA levels were similar (Figure 3b).

4. Discussion

The role of the Sp6 transcription factor, Sp6, in tooth morphogenesis has been demonstrated in the mouse Sp6 knockout model [19,20]. Epfn^-/- mice exhibited supernumerary teeth, deficient enamel, defective cusp and root formation, delayed eruption, and abnormal dentin structure, along with defects in several epithelium-containing organs or appendages, such as skin, hair follicles, and digit formation. Identification of a 2-bp insertional Sp6 mutation in the spontaneous amelogenesis imperfecta rat model with autosomal recessive inheritance [21] further suggested SP6 as a candidate gene for human AI with or without other non-oral defects.

Recently, a missense SP6 mutation was identified in a Caucasian family to cause autosomal dominant hypoplastic AI [18]. The mutation was a 2-bp variant (c.817_818GC>AA) that changed the highly conserved alanine to lysine [p.(Ala273Lys)] in the first of the three zinc finger domains. The mutation was predicted to alter the DNA-binding ability, and the mutant protein was demonstrated to have a reduced binding ability and faster dissociation rate compared to the wild-type when interacting with the AMBN promoter sequence. The clinical phenotype was characterized by generalized hypoplastic AI with an irregular surface on all teeth, and there were no other oral and non-oral defects.

In this study, whole exome sequencing and trio analysis identified a de novo SP6 mutation. Interestingly, the identified mutation was a 2-bp variant at the same nucleotide positions as the previous report [18] but with an AT instead of an AA insertion (c.817_818delinsAT), changing alanine to methionine [p.(Ala273Met)].

There are some differences in phenotype between the proband of this study and the affected individuals from the previous report, even though the same codon was mutated. The proband in this study with the de novo mutation has an unusual root development, taurodontism, and severe hypoplastic AI, while the generalized hypoplastic AI with an irregular enamel surface, representing a mild phenotype, was evident in the previous report. Recently, the Msx2-Sp6-Follistatin pathway, controlling the ameloblast life cycle and amelogenesis, has been demonstrated in a mouse model [22]. In addition, the phenotype of the murine knockout models suggests the involvement of the Sp6 transcription factor in dentin formation. In addition to the reduced DNA binding ability demonstrated in the previous study, the apparently low level of mutant protein expression in this case is likely responsible for the severe dental phenotypes. However, it is not clear that the SP6 mutation examined in this study is solely responsible for the unique root development and taurodontism because there can be other contributing factors. Therefore, further studies are necessary to find the functional role of SP6.

X-linked hypoplastic AI can be caused by mutations in AMELX [23]. Autosomal dominant hypoplastic AI can be caused by mutations in ENAM, COL17A1, and LAMB3 [3,24]. While mutations in AMBN and ACP4 cause non-syndromic autosomal recessive hypoplastic AI [14,17], mutations in FAM20A cause syndromic autosomal recessive hypoplastic AI (enamel-renal syndrome, OMIM#204690) [25]. This study confirmed that the SP6 mutation causes hypoplastic AI in an autosomal-dominant inheritance patterns, in contrast to the mouse phenotype.

In conclusion, we identified a novel de novo mutation in the SP6 gene in a hypoplastic AI family with a peculiar developing root form and taurodontism. This study will expand the mutational spectrum of rare SP6 mutations and expand our understanding of the involvement of SP6 in tooth development.