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Novel Identification of a Four-base-pair Deletion Mutation in PITX2 in a Rieger Syndrome Family
1 Department of Prosthodontics, School of Stomatology, Peking University, 22 Zhong Guan Cun Nan Da Jie, Beijing 100081, People’s Republic of China; and Correspondence: *corresponding author, kqfenghl{at}bjmu.edu.cn
Rieger syndrome is one of the most serious causes of tooth agenesis. Mutations in the PITX2, FOXC1, and PAX6 genes have been associated with Rieger syndrome. We have studied a three-generation Chinese family affected with Rieger syndrome and showing prominent dental abnormalities. Mutational screening and sequence analysis of the PITX2 gene revealed a previously unidentified four-base-pair deletion of nucleotides 717-720 in exon 5 in all affected members. The mutation causes a frame shift after Thr44, the 7th amino acid of the homeo-domain, and introduces a premature stop codon in the gene sequence. This deletion is the first unquestionable loss-of-function mutation, deleting all the functionally important parts of the protein. Our novel discovery indicates that the oligodontia and other phenotypes of Rieger syndrome observed in this family are due to this PITX2 mutation, and these data further support the critical role of PIXT2 in tooth morphogenesis.
Key Words: oligodontia • Rieger syndrome • gene mutation • family
Rieger syndrome (MIM 180500) is an autosomal-dominant disorder characterized by dental hypoplasia, mild craniofacial dysmorphism, ocular anterior chamber anomalies, and umbilical stump abnormalities (Amendt et al., 2000). It is one of the most serious causes of tooth agenesis, and the incidence rate of Rieger syndrome is about 1:200,000 (Gorlin et al., 1976). Rieger syndrome has been genetically linked to mutations on chromosomal loci 4q25, 13q14, 6p25, and 11, the most common of these being 4q25 (Phillips et al., 1996; Semina et al., 1996; Mirzayans et al., 2000; Riise et al., 2001). Positional cloning of the 4q25 region identified the PITX2 gene, which codes for a homeo-domain transcription factor (Semina et al., 1996). A second locus for Rieger syndrome was mapped to chromosome 13q14 (Phillips et al., 1996), but the gene has not yet been identified. A third locus, 6p25, encodes the forkhead-like transcription factor FOXC1 (formerly known as FKHL7), which was first linked to glaucoma phenotypes (Nishimura et al., 1998), and later to patients with Rieger syndrome (Mirzayanse et al., 2000; Kawase et al., 2001). Finally, the locus for Rieger syndrome on chromosome 11 has been associated with small deletions in the pair-like transcription factor gene PAX6 (Riise et al., 2001). All these Rieger-syndrome-associated genes—PITX2, FOXC1, and PAX6—encode transcription factors and have been shown to play important roles in embryonic development (Gage et al., 1999; Kioussi et al., 1999; Kume et al., 2000). Clinical presentations of Rieger syndrome are varied with regard to tooth anomalies, including all or some of the following: missing teeth (hypodontia or anodontia), microdontia, abnormally shaped teeth, and abnormally implanted teeth (Semina et al., 1996). It has been observed that, in older patients, the teeth become brittle and are lost at an early age (Espinoza et al., 2002). The analysis of Rieger syndrome patients provides evidence for PITX2 involvement in tooth development. The expression of Pitx2 is restricted to the dental epithelium during tooth development, and Pitx2 transcripts can be detected as early as Embryonic Day 8.5, well before tooth initiation in mouse models (Mucchielli et al., 1997; St Amand et al., 2000). Post-natal expression of the gene is still detected in relatively undifferentiated epithelial tissue in the tooth germs, and in the later-developing second and third molar anlages (Mucchielli et al., 1997). The molecular basis of tooth anomalies in Rieger syndrome appears to be the inability of mutant PITX2 to activate genes involved in tooth morphogenesis. PITX2 greatly up-regulates the activity of Dlx2, another transcription factor involved in early tooth development (Espinoza et al., 2002). In this study, we analyzed the homeobox region in the PITX2 gene in a three-generation Chinese family with Rieger syndrome. Our mutation analysis revealed a four-base-pair deletion in the PITX2 homeobox which co-segregates the disease in the family.
Pedigree Construction The proband is a 12-year-old boy who had not developed most of his permanent teeth at the time of presentation. Further examination revealed ocular symptoms of Rieger syndrome. His mother also suffered from similar symptoms. Through subsequent interviews, we extended the pedigree to three generations, including 13 individuals, five affected and eight unaffected (Fig. 1  ). The diagnosis of oligodontia was based on clinical examination and panoramic radiographs. The present study was conducted under informed consent of all family members and approved by the Ethics Committee of the Peking University Health Science Center.
PCR-SSCP Analysis Peripheral blood samples were obtained from nine members of the family, including the five patients. Genomic DNA was extracted from these samples by means of a blood DNA mini kit (Watson Biotechnologies, Inc., Shanghai, PRC). We used polymerase chain-reaction (PCR) to amplify the entire homeobox sequence and portions of the non-coding sequence of PITX2 with primers (Fig. 2A ) as described previously (Semina et al., 1996). PCR products were screened for mutations in PITX2 by SSCP analysis. Electrophoresis was performed on the DCodeTM Universal Mutation Detection System (BIO-RAD, Hercules, CA, USA). After being silver-stained according to standard protocols, the gels were visually inspected for potential abnormal variants.
Sequencing PCR fragments were gel-purified by means of an E.Z.N.A.® Gel Extraction kit (Omega Bio-tek, Inc., Doraville, GA, USA) according to the manufacturer’s protocol. Sequencing analyses were performed by ABI Big Dye terminator reagents 2.0 (Applied Biosystems, Foster City, CA, USA) on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). To determine the exact status of mutation, we also cloned PCR fragments of exon 5, amplified from patients I:2 and III:4, into pGEM®-T Easy Vectors (Promega Corporation, Madison, WI, USA). After being transformed into competent E. coli strain TOP10, colonies carrying recombinant plasmid from both individuals were picked, and the plasmid DNA was extracted by means of a MiniBEST Plasmid Purification Kit [TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, People’s Republic of China]. Sequencing analysis was performed with use of the flanking vector primers by the same method described above.
Clinical Diagnosis All affected family members were missing most of their permanent teeth (at least 19) (Table 1 ), though they claimed to have had normal primary dentition as children. Often, due to the lack of permanent teeth pushing through, these individuals retained their deciduous teeth into and through their 30s.
The proband, III:4, was missing his 27 permanent teeth (including wisdom teeth) without a medical history of tooth extraction. Intra-oral examination indicated that most of the teeth left were retained primary teeth. Panoramic radiography showed that the right upper second molar and left lower second molar were underdeveloped. The photograph also showed extensive root resorption of all remaining primary teeth (Fig. 1B  ). The patient had a class III jaw relationship with midface deficiency and mild prognathism. Upon ophthalmic examination, the patient showed evidence of congenital hypoplasia of the anterior chamber of both eyes, including iris hypoplasia, microcornea, and anterior chamber synechiae. The intra-ocular pressure was normal at the time of examination. The medical history of surgical treatment for congenital umbilical hernia after birth fulfilled the final crucial feature for the diagnosis of Rieger syndrome. The clinical details of the family are shown in Table 1.
Mutation Analysis
Sequence analysis of the aberrant fragments showed overlapping peaks after nucleotide 716 of the cDNA (in exon 5), indicating the presence of a frameshift mutation. After subcloning the PCR products from patients I:2 and III:4 and analyzing the sequence of individual clones, we found the mutated allele to have a deletion of 4 bp, ACTT (nucleotide 717-720), that alters the PITX2 reading frame and results in the introduction of a stop codon 39 residues after Thr44 (Fig. 2C
In this paper, we report a novel four-base-pair deletion mutation in PITX2 that is responsible for Rieger syndrome in a three-generation Chinese family. The deletion of ACTT, 717-720, in exon 5 of PITX2 results in a reading frame shift and truncates the PITX2 protein at the 7th amino acid of the homeo-domain of the DNA-binding site (Fig. 2A ). This is the first unquestionable loss-of-function mutation, deleting all the functionally important parts of the protein.
The PITX2 gene is a member of the bicoid-like homeobox family (Semina et al., 1996). The homeobox encodes a 60-amino-acid homeo-domain that binds DNA. The integrity of the homeo-domain is essential for DNA binding and is critical for PITX2’s activity as a transcription factor. So far, 23 different PITX2 mutations have been identified in the patients with Rieger syndrome and other ocular anomalies (Table 2
The dental anomalies in this family are very prominent. Such serious dental dysplasia has not been described in previous reports of Rieger syndrome. In our report, the patients are congenitally missing from 19 to 27 permanent teeth. All of them are missing their front teeth, second premolars, and third molars. Most of their remaining teeth are malformed. This pattern of tooth agenesis shows that the function of PITX2 is seriously defective. Earlier studies have shown that PITX2 mutations in which some function is retained result in milder ocular phenotypes, whereas the mutations in which PITX2 is inactive result in more serious phenotypes (Kozlowski and Walter, 2000). This result suggests that variance in PITX2 activity may underlie different degrees of dental anomalies. Further evidence comes from the DNA binding specificity of PITX2 to the Dlx2 promoter. A phenotypically less severe mutant PITX2 (without tooth anomalies) had a similar DNA binding specificity compared with wild-type PITX2 and transactivated the Dlx2 promoter. In contrast, another mutant PITX2 which presented clinically with the full spectrum of developmental anomalies (including tooth anomalies) was unable to transactivate the Dlx2 promoter (Espinoza et al., 2002). Thus, it is easy to understand why our reported loss-of-function mutation results in the most serious dental anomalies in existing studies. Oligodontia could be linked to several diseases, such as Rieger syndrome, hypohidrotic ectodermal dysplasia, and non-syndromic tooth agenesis. Since many Rieger syndrome patients require dental prosthetic treatment at an early age, we propose that dentists should become more familiar with this disease and its severe outcomes. Our result could be used for pre-natal diagnosis of or mutation screening for Reiger syndrome.
We thank the family members for their willing cooperation and participation, Dr. Dalong Ma for his critical discussion and review of this manuscript, and members of the Peking University Human Disease Genomics Center for their excellent technical assistance. This work was supported by the Special fund for promotion of education, Ministry of Education, P.R. China (Peking University Center for Human Disease Genomics: 2001-10).
3 equal contributors to this paper;  Received for publication January 31, 2003. Revision received June 27, 2003. Accepted for publication September 12, 2003.
Journal of Dental Research, Vol. 82, No. 12,
1008-1012 (2003) This article has been cited by other articles:
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