Novel ITGB6 mutation in autosomal recessive amelogenesis imperfecta.

OBJECTIVE: Hereditary defects in tooth enamel formation, amelogenesis imperfecta (AI), can be non-syndromic or syndromic phenotype. Integrins are signaling proteins that mediate cell-cell and cell-extracellular matrix communication, and their involvement in tooth development is well known. The purposes of this study were to identify genetic cause of an AI family and molecular pathogenesis underlying defective enamel formation.
MATERIALS AND METHODS: We recruited a Turkish family with isolated AI and performed mutational analyses to clarify the underlying molecular genetic etiology.
RESULTS: Autozygosity mapping and exome sequencing identified a novel homozygous ITGB6 transversion mutation in exon 4 (c.517G>C, p.Gly173Arg). The glycine at this position in the middle of the βI-domain is conserved among a wide range of vertebrate orthologs and human paralogs. Clinically, the enamel was generally thin and pitted with pigmentation. Thicker enamel was noted at the cervical area of the molars.
CONCLUSIONS: In this study, we identified a novel homozygous ITGB6 mutation causing isolated AI, and this advances the understanding of normal and pathologic enamel development.

Introduction

A series of ectomesenchymal interactions are involved in the development of teeth (Thesleff, 2003). Once the odontoblasts secrete the initial dentin matrix, enamel begins to form. The process of enamel formation (amelogenesis) can be classified into presecretory, secretory, transition, and maturation stages. A genetic defect affecting any stage of amelogenesis can cause stage-specific enamel defects (Hu et al, 2007). The affected enamel can be one or a mixed form of the hypoplastic, hypocalcified, or hypomatured type (Seymen et al, 2014b).

Amelogenesis imperfecta (AI) is a collection of hereditary diseases affecting tooth enamel formation (Witkop, 1988). AI can be an isolated form without any other non-oral symptoms or a phenotype of syndromic conditions, such as enamel-renal syndrome (OMIM #204690; FAM20A) (Jaureguiberry et al, 2012; Wang et al, 2013a) and Jalili syndrome (OMIM #217080; CNNM4) (Parry et al, 2009). To date, more than 10 genes have been identified as being involved in the pathogenesis of AI.

Genetic studies on the pathogenesis of AI have been focused on the genes encoding enamel matrix proteins, and mutations have been identified in the amelogenin (AMELX) (Lagerstrom et al, 1991; Cho et al, 2014), enamelin (ENAM) (Rajpar et al, 2001; Seymen et al, 2014a), ameloblastin (AMBN) (Poulter et al, 2014b), enamelysin (MMP20) (Kim et al, 2005), and kallikrein 4 (KLK4) genes (Hart et al, 2004; Wang et al, 2013b). In addition, mutations in novel genes, such as family with sequence similarity 83 member H (FAM83H) (Kim et al, 2008), chromosome 4 open reading frame 26 (C4orf26) (Parry et al, 2012), WD repeat-containing protein 72 (WDR72) (El-Sayed et al, 2010; Lee et al, 2010), and solute carrier family 24 member 4 (SLC24A4) genes (Parry et al, 2013; Seymen et al, 2014b), have been identified by locus mapping and/or whole-exome sequencing.

Junctional epidermolysis bullosa (JEB) is a rare hereditary skin disease featuring blister formation and AI in an autosomal recessive hereditary pattern (Masunaga, 2006). JEB has been known to be caused by mutations in the genes encoding hemidesmosome-anchoring complexes, such as laminin alpha 3 (LAMA3), laminin beta 3 (LAMB3), laminin gamma 2 (LAMC2), collagen type XVII alpha 1 (COL17A1), integrin beta 4 (ITGB4), and integrin alpha 6 (ITGA6) (Intong and Murrell, 2012). Carriers usually have no disease phenotype; however, rarely, heterozygous conditions can cause AI with no or very mild skin fragility in an autosomal dominant mode, probably due to a dominant-negative effect of a defective allele (Kim et al, 2013).

Recently, two cases of homozygous mutations and one case of compound heterozygous mutations in the integrin beta 6 (ITGB6) gene have been reported to cause AI, and its stage-specific expression in ameloblast differentiation has been shown (Poulter et al, 2014a; Wang et al, 2014). In this report, we recruited a consanguineous family with a proband having hypoplastic AI and identified a novel homozygous ITGB6 mutation.

Methods

Enrollment of human subjects

A consanguineous Turkish family having hypoplastic AI was recruited for genetic studies. The study protocol was reviewed and approved by the Institution Review Board at Seoul National University Dental Hospital and by the University of Istanbul. Clinical and radiological examinations were performed, and blood samples were collected with the understanding and written consent of each participant according to the Declaration of Helsinki.

Autozygosity mapping

DNA was isolated from peripheral whole blood of the participating family members using the QuickGene DNA whole blood kit S with the QuickGene-Mini80 equipment (Fujifilm, Tokyo, Japan). All family members (V:1, V:2, VI:1, and VI:2) (Figure1) were genotyped with the Affymetrix Genome-Wide Human SNP array 6.0 by Macrogen (Seoul, Korea). The annotated SNP files were analyzed with HomozygosityMapper (http://www.homozygositymapper.org/) to identify the region of homozygosity in the proband.

Figure 1

Pedigree, clinical photographs, and panoramic radiograph of the family. (a) Pedigree of the family. Consanguineous marriages are indicated with double lines. Family members who participated in this study are indicated under the symbol (V:1, V:2, VI:1, and VI:2). Proband is indicated with black arrow. (b) Frontal clinical photograph of the proband at age 8. (c) Frontal clinical photograph of the proband at age 10. Maxillary and mandibular anterior permanent teeth are restored with direct resin composite. (d) Maxillary clinical photograph of the proband at age 10. (e) Mandibular clinical photograph of the proband at age 10. Enamel is generally thin with some area of pitted pigmentation. Thicker enamel can be seen in the cervical part of the molar teeth. (f) Panoramic radiograph of the proband at age 8. The reduced thickness and radiodensity of the enamel can be seen in the developing permanent teeth.

Whole-exome sequencing

Whole-exome sequencing was performed with the DNA sample of the proband after exome capturing with the NimbleGen exome capture reagent. Of 75-bp paired-end sequencing reads were obtained with Illumina HiSeq 2000 (Yale Center for Mendelian Genomics, West Haven, CT, USA). Sequencing reads were aligned to the NCBI human reference genome (NCBI build 37.2, hg19), and the sequence variations were annotated with dbSNP build 138.

In silico analysis

Annotated variants with low sequencing quality were filtered first, and those in the dbSNP 138 were excluded. Remaining variants were analyzed in silico with Align GVGD (http://agvgd.iarc.fr/) (Tavtigian et al, 2006), SIFT (http://sift.jcvi.org/) (Ng and Henikoff, 2003), Mutation Taster (http://www.mutationtaster.org/) (Schwarz et al, 2010), and PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) (Adzhubei et al, 2010). An ITGB6 variant was further analyzed with the Provean (http://provean.jcvi.org/) (Choi et al, 2012) and MutPred (http://mutpred.mutdb.org/) (Li et al, 2009) programs.

Polymerase chain reaction and sequencing

The identified variation in the ITGB6 gene was confirmed with Sanger sequencing, and segregation within the family was confirmed with exon 4 primers (sense: 5′-TGAAAGAATTTCATGGGTTGG, antisense: 5′-GGCCTCTGAGAGAACTGCTG). Polymerase Chain Reaction (PCR) amplifications were performed with the HiPi DNA polymerase premix (Elpis Biotech, Taejeon, Korea), and PCR amplification products were purified with a PCR Purification Kit and protocol (Elpis Biotech). DNA sequencing was performed at a DNA sequencing center (Macrogen).

Results

The proband was an 8-year-old girl from a consanguineous marriage, who presented with hypoplastic enamel and thermal sensitivity (Figure1). The enamel was generally thin, but thicker enamel was noted at the cervical area of the molars. Enamel surfaces had also pitted areas with pigmentation. A panoramic radiograph showed a certain amount of reduction in thickness of the enamel in the developing teeth. The thin enamel may be the result of excessive wear due to the microscopically less mineralized enamel. Reduction in the radiopacity of the enamel was shown in the panoramic radiographic examination.

The array data were first analyzed for the pathologic copy number variation (CNV), but failed to identify any possible disease-causing CNV (data not shown). Homozygosity mapping revealed 18 regions of loss of heterozygosity (Figure2). The exome data of the proband were annotated with the dbSNP build 138. Quality filtering and SNP filtering resulted in six candidate homozygous variants (Table 1). In silico analyses with Align GVGD, SIFT, Mutation Taster, and PolyPhen-2 consistently indicated that the ITGB6 variant would be deleterious. The ITGB6 variant was further analyzed with the Provean and MutPred programs, and both results also indicated a deleterious effect with significant scores (Table 2).

Figure 2

Mutational analysis. Autozygosity mapping identified candidate regions. Among the regions, a long region of loss of heterozygosity in chromosome 2 is drawn below. The region locations and genes in the regions are shown. Sanger sequencing chromatograms of the family members are shown. Nucleotide sequences are shown above the chromatograms. A red arrow indicates the mutation (c.517G>C, p.Gly173Arg). S indicates G or C nucleotides.

Table 1

In silico analysis of the filtered variants

Chr	Gene	Changes	Align GVGD	SIFT	Mutation Taster	PolyPhen-2
chr15	ATP10A	NM_024490:c.T2977G:p.F993V	Class C0	Tolerated (score: 0.53)	Polymorphism	Benign (score = 0.003)
chr2	MARCH7	NM_001282806:c.A959G:p.E320G	Class C0	Tolerated (score: 0.06)	Disease causing	Possibly damaging (score = 0.953)
chr2	ITGB6	NM_000888:c.G517C:p.G173R	Class C65	Deleterious (score: 0)	Disease causing	Probably damaging (score = 1.000)
chr20	TAF4	NM_003185:c.A397T:p.S133C	Class C0	Tolerated (score: 0.11)	Polymorphism	Possibly damaging (score = 0.953)
chr20	LAMA5	NM_005560:c.C1957T:p.R653C	Class C0	Tolerated (score: 0.07)	Disease causing	Benign (score = 0.238)
chr8	CSPP1	NM_024790:c.A3463G:p.S1155G	Class C0	Tolerated (score: 0.3)	Polymorphism	Benign (score = 0.000)

Table 2

In silico analysis of the ITGB6 variant

Mutation	PolyPhen2	MutationTaster	SIFT	PROVEAN	MutPred
c.G517C	Score = 1.000	Probability = 0.999	Score = 0	Score = −7.731	Score = 0.931
p.Gly173Arg	Probably damaging	Disease causing	Damaging	Deleterious	Gain of methylation at G173 (P = 0.0136)

URLs: Align GVGD, http://agvgd.iarc.fr/ (Tavtigian et al, 2006); PolyPhen2, http://genetics.bwh.harvard.edu/pph2/ (Adzhubei et al, 2010); Mutationtaster, http://www.mutationtaster.org/ (Schwarz et al, 2010); SIFT, http://sift.jcvi.org/ (Ng and Henikoff, 2003); PROVEAN, http://provean.jcvi.org/ (Choi et al, 2012); MutPred, http://mutpred.mutdb.org/ (Li et al, 2009).

Sanger sequencing confirmed the existence and cosegregation of the ITGB6 variant with the disease within the family members. Additionally, this variant was not found in the NHLBI exome variant server (http://evs.gs.washington.edu/EVS/) and the 1000 Genome database (http://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/). The mutation was a transversion of a guanine to a cytosine (NM_000888.4; c.517G>C), resulting in a change of glycine to arginine at codon position 173 (NP_000879.2; p.Gly173Arg). Glycine at this position was completely conserved among a wide range of vertebrate orthologs (Figure3). Sequence alignment between all human ITGB gene family members (ITGB1∼8) also showed complete conservation of Glycine at this position.

Figure 3

Sequences alignments of part of the ITGB6 gene. Conservation of the Gly173 is indicted with red color in the ITGB6 vertebrate orthologs and human paralogs.

Discussion

Integrins are heterodimeric cell-surface receptors that contain α and β subunits (Hynes, 2002). Both subunits are type I membrane proteins and non-covalently associated with form heterodimers. At least 24 integrin receptors have been identified that are assembled from the 18 α and 8 β subunits in mammals. Integrins have diverse roles in various biological processes by mediating cell–cell, cell–extracellular matrix, and cell–pathogen interactions. Integrins can transmit signals in both directions across the membrane.

Defective integrin function has been shown to be related to human genetic diseases. Defects in αIIbβ3 integrin (the major platelet integrin) by mutations in the genes encoding the integrin αIIb (ITGA2b) and integrin β3 subunits (ITGB3) cause a bleeding disorder known as Glanzmann thrombasthenia (Kato, 1997). Mutations in the integrin β2 subunit (ITGB2) cause leukocyte adhesion deficiency, which leads to leukocytosis and early death from a defective host defense (Etzioni et al, 1999). Mutations in integrin β4 (ITGB4) and integrin α6 (ITGA6) cause JEB with pyloric atresia (Pulkkinen and Uitto, 1999).

The functional roles of several integrins during tooth development have been elucidated. The involvement of integrin αvβ5 has been suggested in epithelial–mesenchymal interactions during tooth development, and expression of integrin α6, β1, and β4 subunits has been shown to be involved in the developing tooth epithelium (Salmivirta et al, 1996). An enamel defect is indeed a syndromic phenotype of patients with JEB and pyloric atresia. Recent findings in an integrin β3 subunit knockout mouse model revealed that iron transport is defective due to reduced expression of Slc11a2 and Slc40a1, resulting in a loss of pigmentation in the lower incisors (Yoshida et al, 2012).

Itgb6 null mice were recently reported to cause enamel malformation that resulted in hypomaturation lacking normal enamel rod structures and severe attrition resembling human hypomaturation AI (Mohazab et al, 2013). An immunohistochemical study showed that the expression of ITGB6 was localized to the distal membrane of differentiating ameloblasts and pre-ameloblasts and then internalized by the secretory stage ameloblasts (Wang et al, 2014). However, the strongest expression appeared in the maturation stage ameloblasts associated with ameloblast modulation.

The head of the large extracellular domain of integrin heterodimers is composed of a propeller domain from the α subunit, and a βI-domain and hybrid domain from the β subunit (Xiong et al, 2001). The mutation identified in this study would change a glycine in the middle of the βI-domain, which is conserved among a wide range of vertebrate orthologs and human paralogs, to an arginine (p.Gly173Arg). This mutation changes a nonpolar amino acid with a neutral side chain charge (hydropathy index -0.4) to a polar amino acid with a positive side chain charge (hydropathy index -4.5); therefore, it is likely to introduce a pathologic conformational change that results in the disruption of the interaction with the integrin αv subunit and the subsequent function of the integrin heterodimer during tooth development.

Determining the exact clinical phenotype in humans is difficult sometimes. Mutant mice lacking Itgb6 exhibited less mineralized enamel (Mohazab et al, 2013). A homozygous mutation identified in a Pakistan family (p.Pro196Thr) resulted in pitted hypomineralized AI (Poulter et al, 2014a). However, clinical phenotypes related to the mutations (p.Arg616* and p.[Ala143Thr];[His275Gln]) identified in two Hispanic families were generalized hypoplastic AI (Wang et al, 2014). Interestingly, the mutation identified in our Turkish family (p.Gly173Arg) resulted in an in-between phenotype of the pitted hypoplastic enamel with hypomineralization. Because AI cases caused by ITGB6 mutations are very rare, to date, there have been only a limited number of affected individuals (three affected individuals from three families and three affected individuals from a single family) (Table 3). Given the genetic heterogeneity of the human population, unlike the mouse study with the same genetic background, phenotypic variations could be natural.

Table 3

Disease-causing mutations in the ITGB6 gene

Location	cDNA	Protein	Mode of inheritance	References
Exon 4	c.427G>A	p.Ala143Thr	Paternal	Wang et al (2014)
Exon 4	c.517G>C	p.Gly173Arg	Homo	This report
Exon 4	c.586C>A	p.Pro196Thr	Homo	Poulter et al (2014a)
Exon 6	c.825T>A	p.His275Gln	Maternal	Wang et al (2014)
Exon 11	c.1846C > T	p.Arg616∗	Homo	Wang et al (2014)

Sequences based on the reference sequence for mRNA (NM_000888.4) and protein (NP_000879.2), where the A of the ATG translation initiation codon is nucleotide 1.

In summary, we identified a novel homozygous missense mutation, changing an absolutely conserved amino acid in the middle of the βI-domain of the integrin β subunit, in a consanguineous Turkish family. We believe that this finding will extend the mutational spectrum of the ITGB6 gene and broaden the understanding of normal and pathologic tooth development.