Study Of A Family With Marfan Syndrome Caused By A Novel Mutation Of Fibrillin-1Gene

Objiective:Marfan syndrome (MFS), an autosomal dominant inherited connective tissue disease, has variable clinical manifestations typically affecting the cardiovascular, ocular and skeletal systems. Two causative genes for MFS have been identified so far:Fibrillin-1(FBN1) and TGFBR2gene. This study aims to characterize the clinical features in a Chinese family with autosomal dominant MFS, to map the disease locus by exclusive linkage analysis, to indentify the disease causing gene by mutation screening, and to correlate the mutation genotype and clinical phenotype in the family. Method:A family with autosomal dominant MFS was recruited at clinic. The detailed clinical examinations of cardiovascular system, including color doppler ultrasonography, measurement of aortic root diameter and observation of valve prolapsed, were performed and documented on each patient. All patients were also subjected to other systemic examinations including the measurement of height, skeletal features, skin extensibility, assessment of best correct visual acuity, slit-lamp and direct fundus examinations. The aortic tissue from the proband was collected during surgery, and further sectioned for histology study. Peripheral blood samples were collected from all participants. In addition, peripheral blood samples were collected fiom100normal Chinese volunteers with neither history of MFS nor related fibrillinopathy and used as normal controls in the mutation study. Written informed consent was obtained for genomic analysis and clinical investigations from all participants, and the Ethic Committee of Tianjin Medical University approved this study. Genomic DNA was extracted from peripheral blood leukocytes using DNA Isolation Kits for Mammalian Blood (Roche Biochemical, Inc.) and used for genotyping in all individuals of the MFS family. Because only two genetic loci have been identified in MFS, our initial genetic study was focused on linkage analysis with markers linked to known loci for MFS:FBN1(D15S659, D15S992, D15S1016and D15S648), TGFBR2(D3S2336, D3S2335, D3S1283, D3S3727and D3S2432). Amplification of these microsatellite markers by polymerase chain reaction (PCR) was performed using primers labelled by Fam, Tet or Hex and experimental conditions as previously described. The PCR products were appropriately pooled according to allele size and labelling, mixed with GeneScanTM-500TAMRATM standard (ABI), denatured, loaded onto6%standard denaturing polyacrylamide gels and run in ABI377XL sequencer for fluorescent detection. Genotyping data were collected using GeneScan Analysis3.1and analysed using the Genotyper2.0software package (Perkin Elmer). Linkage analyses were carried out by calculating Multipoint LOD scores using the LINKAGE software package of Simwork2, Version3.35. MFS in the families was modelled as an autosomal dominant trait with a disease-allele frequency of0.0001and a penetrance of99%. The allele frequencies for each marker were assumed to be equal, as well as the recombination frequencies in males and females. In the calculations, individuals Ⅱ:1, Ⅲ:1,2,7,8,9, Ⅳ:1,2,3,4of kindred MF1were scored as affected and all other members as unaffected. Family and haplotype data were generated using Cyrillic (version2.1) software. Results:All affected individuals were diagnosed as MFS syndrome. After exclusion of the TGFBR2locus by linkage analysis, significant linkage was found with markers of the FBN1locus in the15q21.1region in the family. By direct Sanger sequencing of FBN1gene, we further identified a novel heterozygous base substitution (C→A) was detected at nucleotide position946of exon7, which creates a premature stop codon (TGA) instead of a cysteine (TGC) at codon271(C271X). The nonsense mutation was proved to co-segregate with the disease phenotypes in the family, and were absent in200chromosomes from100unrelated control individuals, probably excluding the possibility of a rare polymorphism. Conclusion:The mutations of FBN1are associated with a broad spectrum of manifestations, of which the clinical severities range from sole connective tissue disease to embryonic lethal. Substitutions of the conserved cysteine residue, the most common mutations, often cause the rather mild phenotype of cardiovascular system. However, ocular and skeletal system are commonly involved in patients with such mutations. The novel nonsense mutation reported herein generates a PTC codon and the consequent truncated mRNA, which is likely to be degraded by nonsense mediated decay. Thus, the PTC mutation of FBNl gene is considered as server, and is presumably related with the server phenotype, specifically the cardiovascular malformation, in the family.