Analysis Of Disease Gene Mutation And Function In A Patient With Axenfeld-Rieger Syndrome

Axenfeld–Rieger syndrome (ARS) is a rare autosomal dominant inherited disorder withan estimated incident rate of1:20000. It is characterized by mild craniofacial dysmorphism,ocular and dental anomalies, redundant periumblical skin.50percent of ARS patients haveglaucoma. Pituitary abnormalities, hearing loss, cardiac defects, hypospadias, growthretardation and mental retardation may also be found in some ARS patients. So far, twoknown genes PITX2on chromosome4q25and FOXC1on6p25were associated with ARS.One family with ARS was mapped to another locus on chromosome13q14. However, itsdisease-causing gene has not been identified yet.In this study, an ARS patient has been identified and characterized. The patient showedocular anomalies, missing teeth, nails growth arrest and redundant periumblical skin.Mutational analysis of all exons and exon-intron boundaries of PITX2were carried out usingdirect DNA sequence analysis. A>G substitution at position-11of intron4(IVS4-11A>G) ofPITX2was found in the patient. But this substitution was not identified in the mother, auntand brother of the patient. The IVS4-11A>G may be a de novo variation of the patient.Bioinformatics software predicted that the variation may generate a new acceptor site. Todetermine the consequence of IVS4-11A>G on the splicing of PITX2transcripts, expressionvectors containing normal and mutant PITX2minigenes were constructed and transfectedinto Hela cell, respectively. RT-PCR amplification of PITX2transcripts isolated from thecells transfected with the wild-type minigene generated a major product consistent withnormal splicing of intron4, whereas the IVS4-11A>G allele also produced one similar towild type product in patient. Sequence analysis showed IVS4-11A>G induced the acceptorsite of intron4shifted exclusively to the newly created "AG" dinucleotide11bp upstream ofthe authentic one. The abnormal splicing could lead to an insertion of10bases of intron4sequence into the mature RNA, resulted in a new protein containing246residues of PITX2.The mutant protein lacked functional homeodomain (HD) and OAR (otp aristaless and rax)domain, may disturb the nuclear localization signal (NLS) of PITX2. The wild type andmutant PITX2-pEGFP-C1vectors were constructed and transfected into Hela cells. Analysis of subcellular localization showed that wild type protein located only in nucleus, whearasemutant protein mainly located in cytoplasm. Moreover, mutant protein was mainlyco-localized with endoplasmic reticulum, and some co-localized with Golgi body.PITX2gene is widely expressed in heart, lung, brain, tooth and pituitary of vertebrata.PITX2is downstream of Nodal-Shh pathway and Wnt-Frizzled-β-catenin pathway,regulating the embryo development. IVS4-11A>G mutation resulted in a truncated proteinwhich lacked functional HD and OAR domain. The mutant protein is mainly located incytoplasm, and may lose DNA binding and transactivation abilities. DKK2and DLX2are thedownstream target genes of PITX2. Dysregulation of DKK2and DLX2could result inabnormal phenotypes of ARS patients.In conclusion, the results show the splice mutation IVS4-11A>G of PITX2gene is thereal cause of ARS.