Mutation Screening In Families With Two Rare Diseases By Candidate Gene Approach

Objective : Marfan syndrome is a dominant hereditary connective tissue disease,with an incidence ranging from 1/3000-1/5000[1],multiple systems and organs could be damaged,mainly involving the skeleton,cardiovascular and ocular visual system.There is no particular treatment measure for Marfan syndrom,surgical and medical treatments are approached based on the symptoms.A large number of studies confirmed that the mutation of human fibrillin-1 gene(FBN1)plays an important role in the pathogenesis of Marfan syndrome.Congenital aniridia is a rare autosomal disease,with an incidence ranging from 1/64,000 to 1/96,000 in different ethnic groups[2-4].The most prominent clinical feature is the degeneration of the iris tissue,and the root of which is usually seen under gonioscope.The mutation of the paired box6 gene(PAX6)could be usually detected in most patients.In this study,FBN1 gene and PAX6 gene were selected as candidate genes for these two diseases.The mutation screening is aimed to explore the molecular pathogenesis of these two rare diseases,and help to promote the development of genetic counseling and prenatal diagnosis.Methods: Two families with Marfan syndrome and one family with congenital aniridia were recruited in our study,which were named MFS-01,MFS-02 and CA-01.683 normal controls were recruited at the same time.The pedigree diagram were finished after medical history taking.All participants of families with Marfan syndrome underwent complete physical,cardiovascular and ophthalmologic examinations.All members in the family with congenital aniridia underwent complete and comprehensive clinical and ophthalmological examinations,including visual acuity test,anterior segment examination,intraocular pressure measurement,fundus examination,and orthoptic evaluation,as well as the examination of physical malformations and neurological deficits.5-10 ml venous blood was collected to extract genomic DNA after obtaining informed consent.Primer 3was used to design the primers,and the sequences of FBN1 and PAX6 were downloaded from NCB1 database.The whole coding regions of FBN1 and PAX6 were amplified by polymerase chain reaction(PCR),amplified PCR products were purified and sequenced directly with an Automated Genetic Analysis system 3730.SIFT and Poly Phen-2 were used to predict the possible structural and functional changes of the protein.Results: A male patient in MFS-01 pedigree was confirmed,who has many clinical manifestations mainly including bilateral absence of crystal,vitreous turbidity,aortic sinus dilatation,tall and thin body,slender fingers and toes.A novel heterozygous mutation c.3998 G>A(p.C1333Y),was found in exon 32 of the child patient,whereas unaffected members and another 683 normal controls did not carry this mutation;Two patients were confirmed in MFS-02,who were a pair of father and daughter,with crystal ectopic,strabismus,skeletal system and other similar clinical manifestations.Besides,the father was with a history of aortic dissection,and daughter suffered from the aortic root dilatation.A novel heterozygous mutation c.1708 T>G(p.C570G)was identified in exon 14 of the two patients,whereas unaffected members and another 683 normal controls did not carry this mutation.Four patients were diagnosed as congenital aniridia in the CA-01 pedigree.The main clinical manifestations were absence of the iris,low vision,nystagmus,cataracts,etc.A novel heterozygous mutation c.275 G>A(p.R92Q)was identified in exon 6 of four patients,whereas unaffected members and another 683 normal controls did not carry this mutation.This substitution of amino acid in the FBN1 and PAX6 protein was predicted to be damaging by SIFT and Polyohen-2.Conclusions: c.3998G>A(p.C1333Y)and c.1708 T>G(p.C570G)mutation in FBN1 may be the cause of Marfan syndrome,and c.275 G>A(p.R92Q)mutation may be the cause of congenital aniridia.The novel mutations found in these three families have not been reported at home and abroad.Our study enriches the mutation spectrum of FBNI and PAX6,which may help to understand the molecular pathogenesis and promote the development of prenatal diagnosis and genetic counseling for MFS and congenital aniridia.