Mutation Analysis Of Forkhead Transcriptional Factor 2 (FOXL2) In A Five-generation Chinese Family With BPES Type Ⅰ

Blepharophimosis-ptosis-epicanthus inversus syndrome(BPES) is a rare eye genetic disorder, affected 1 in 100 000 people. Classic BPES is a complex eyelid malformation invariably characterized by three major features: blepharophimosis, ptosis, and epicanthus inversus, Other manifestations associated with BPES include mental retardation, growth retardation, heart diseases, microcephaly and low-set ears. Two types of blepharophimosis syndrome have been described: BPES type 1 and type II. Type I BPES is an autosomal dominant disorder in which premature ovarian failure (POF) is co-inherited with abnormalities of the eyelids. The infertility is sex-limited, the affected males being fertile and transmitting the trait to the next generation. Both female and male affected individuals show typical facies with characteristic eyelid dysplasia, drooping eyelids and a tiny skin fold running inward and upward from the lower lid. But in type II BPES families, affected males and females are both fertile~1. Linkage analysis and chromosome aberrations in affected patients have indicated that the FOXL2 gene located at 3q23 is responsible for both BPES type I and II.FOXL2 gene is a single exon gene and encode a 2.745 kb transcription factor. The transcript codes for a putative protein of 376 amino acids, which includes a 101-aminoacid DNA-binding fokhead domain from amino acid position 52 to 152. Downstream of the forkhead domain is an alanine rich domain, consisting of 14 alanines from aminoacid position 221 to 234,which is believed to be responsible for transcriptional repression activity~6. Foxl2 gene is the first human autosomal gene in which dominant mutations have been implicated in ovarian maintenance and differentiation.Up to now, there is no effected way to cure the infertilitas feminis caused by BPES type I. use the method of plastic operation can rectification the ophthalmic and facial malformation of BPES patients, and improve their appearance. But BPES patients usually associated with visual disorder and even with other features of systemic disease, which make the patients and their family suffering more distress of body, spirit and economy. Otherwise, the clinical manifestation of BPES is various, and make it difficult for clinical diagnosis. According to the Mendelian genetic rule, the recurrence ring of the off-spring is 50%, So it is significant to carry out genetic counseling on the BPES patients. At present, the methods for prenatal diagnosis are cytogenetics examinations and gene analysis. If the patient has an abnormal chromosome caryogram, cytogenetics examinations could be done for prenatal diagnosis, but if the patient has a normal chromosome caryogram, the gene analysis is the only way. Therefore, the mutation analysis of FOXL2 is the most impotant and the bascal way for the prenatal of BPES.ObjectiveThe purpose of this study is to screen the mutation in the FOXL2 gene in a five-generation Chinese family with BPES type I.Methods1. Chromosme karyotype anlysis were carried out on IV8 and III3 of the family.2. Then the peripheral bolld samples were collected from the affected individuals of the family and healthy indivials as controls, and genomic DNA were isolated using salt fractionation, then the purification and quantitation of genonic DNA were carried out.3. Four overlapping sets of primers were used to amplify the FOXL2 coding region. 4. Afterwards, directly sequencing and polyacrylamide gel electrophoresis (PAGE) were carried out for mutation detection in affected individuals of the family.Results1. Pedigree analysis shows that BPES segregated in this family as an autosomal dominant and the family were defined as the BPES type 1.2. Chromosme karyotype anlysis on IV8 and III3 of the family shows the normal male and femal karyontype as 46,XY and 46,XX.3. Directly sequencing indicated that the BPES phenotype in this family was caused by a 17 bp duplication (1080-1096dup17).4. The images of PAGE exhibited that the affected individuals of the family are heterozygote at FOXL2 CD and EF region.Conclusion1. Direct sequencing revealed a 17bp duplication (1080-1096dup17) in FOXL2 gene of the affected individuals, however, the FOXL2 gene coding region of the healthy individuals were coincidence with the Genebank.2. The 17 bp duplication (1080-1096dup17) we found in this family would cause a frame shift in the reading frame after codon 287, resulting in a truncated protein ending at codon 361.3. Our reaserch report the 1080-1096dup17 mution first time in Chinese people, which can occur in both type of BPES patients, so we suggest that this mutation is an independent origin and it is a mutational hotspot of this disease.