Mutation Screening Of FBN1 In Patients With Ectopia Lentis And Marfanoid Habitus And Related Gene Chip Designing And Verification

The purpose of this study is to systemically analyze the clinical manifestations,and screen the mutations of MFS candidate gene—the FBN1 gene,in the patients with predominant EL and Marfanoid habitus,which will provide further information about genotype-phenotype correlation in MFS.On the other hand,we aim to design a kind of gene chip specific to Marfan syndrome for prenatal diagnosis,which may serve the family planning and the society.Partâ… Ocular Manifestation of the Patients with Ectopia Lentis and Marfanoid HabitusIn this study,10 probands with EL and Marfanoid habitus were collected as well as available family members.After informed consent was obtained,all the affected patients underwent complete physical,ophthalmic,and cardiovascular examinations. The results were summarized in a table for further analysis.Phenotypes of 14 eyes with subluxated lens were available and recorded by slip lamp photography.We found that 9 of the 14 eyes(64.29%) suffered from superior subluxation.A significant smaller(P＜0.0001) mean keratometry(38.66±1.55 D) was noticed in adult EL patients when compared to normal subjects(42.19±1.44 D).On the other hand,the axial length showed a significant increment(P=0.040) in adult EL patients(24.47±2.22 mm) when compared to the control group(23.06±0.86 mm).In the child EL patients,there was a decrease trend in mean keratometry(P=0.101) and an increment trend in axial length(P=0.131) though significant changes could not be found.The above data suggested that the sign of flat cornea might qualify for major criterion for diagnosis of adult MFS.In addition,a relative higher frequency of atrial septal defect(ASD) was noticed in this study,which supported ASD as one of the cardiovascular manifestations for MFS.Partâ…¡Mutation Screening of the FBN1 Gene in the Patients with Ectopia Lentis and Marfanoid HabitusAfter informed consent in accordance with the Zhejiang University Institutional Review Board approval was obtained,blood specimens(5 ml) were collected in EDTA from the patients and available family members.Genomic DNA was extracted from leukocytes of peripheral blood.Then we designed 66 pair primers to amplify every 65 exon of the fibrillin-1 gene(FBN1) as well as the intron-exon boundaries.In order to make the screening more effective,we created priority for each exon.Three novel mutations,c.203G＞T in exon 2,c.502T＞C in exon 5,and c.2096G＞C in exon 16 as well as four known mutations,c.364C＞T in exon 4,c.1633C＞T in exon 13,c.1879C＞T in exon 15,and c.4588C＞T in exon37,were identified in FBN1.None of the seven mutations were detected in the 50 unrelated control subjects with no diagnostic features of MFS.Up to date,genotype-phenotype relationship was still unclear.But we noticed some rules for that.Cysteine substitutions were detected in all the patients with predominant EL in our study,suggesting that cysteine residues may play an important role in suspensory ligaments formation or development.FBN1 variants in patients with predominant EL were clustered in the first 15 exons of the gene.The recurrence of mutations resulting in arginine to cysteine substitutions is significantly higher(P＜0.001,Mann-Whitney U test) than other types of mutations in UMD-FBN1 database.In addition,we reported the first case with FBN1 mutation in the NH₂-terminal domains of fibrillin-1,characterized by EL and myopia in the ocular system,pectus carinatum, arachnodactyly,and dental crowding in the skeletal system whereas neither aortic dilatation nor dissection was shown in the echocardiogram.Partâ…¢Designing and Verification of the Gene Chip Specific to Marfan SyndromeBased on the mutations we detected,we designed a kind of gene chip specific to Marfan syndrome for prenatal diagnosis.Both single nucleotide extension technology with fluorescence labelling and gene chip array were applied to design the gene chip. Six kinds of genomic DNA samples were used to verify the effect of the gene chip, which showed a 100%(6/6) detection rate.Two pair of genomic DNA samples from the proband and her affected mother were simultaneously used to verify the effect of the gene chip,which showed a good repeatability.The gene chip could concurrently serve the 6 families for prenatal diagnosis when the affected want to have a baby someday in the future.Conclusions:1.We systemically analyzed the clinical manifestations of MFS patients in ten Chinese families.It was the first report to categorize the patients into two groups according to age before statistically analysis in MFS patients.The comeas were confirmed to be flattened in adult MFS patients,suggesting that the sign of flat cornea might qualify for major criterion for diagnosis of adult MFS.2.Three novel mutations,c.203G＞T(p.Cys68Phe),c.502T＞C(p.Cys168Arg),and c.2096G＞C(p.Cys699Ser) as well as four known mutations,c.364C＞T(p.Arg122Cys), c.1633C＞T(p.Arg545Cys),c.1879C＞T(p.Arg627Cys),and c.4588C＞T (p.Arg1530Cys),were identified in FBN1.3.According to genotype-phenotype correlation analysis,we noticed that cysteine substitutions were closely related to EL and may play an important role in suspensory ligaments formation or development.FBN1 variants in patients with predominant EL were clustered in the first 15 exons of the gene.The recurrence of mutations resulting in arginine to cysteine substitutions is significantly higher than any other types of mutations.In addition,we reported the first case with FBN1 mutation in the NH₂-terminal domains of fibrillin-1.4.We designed a kind of gene chip specific to MFS for prenatal diagnosis,with high detection rate and repeatability.Although this gene chip could only concurrently serve 6 families for prenatal diagnosis,it verified the feasibility of a high-flux gene chip for different kinds of heredity diseases,multiple families and multiple mutation sites.