Twelve novel FBN1 mutations in Marfan syndrome and Marfan related phenotypes test the feasibility of FBN1 mutation testing in clinical practice.

Marfan syndrome (MFS) is one of the major heritable disorders of connective tissue with a prevalence of between 1 in 5-10 000. 2 It is characterised by features in the cardiovascular, ocular, and musculoskeletal systems and the Ghent criteria form a useful framework for its diagnosis. Mutations in FBN1 encoding the extracellular matrix protein fibrillin-1 classically cause MFS. Fibrillin-1, comprising multiple repeating subunits, of which the most common is the calcium binding epidermal growth factor (cbEGF) domain, is a key component of 10-12 nm microfibrils. Mutation detection has not been performed routinely in MFS because of the size and complexity of FBN1, which contains 65 exons extending over 200 kb of genomic DNA. 6 Mutations are nearly always specific to each family and to date ∼300 mutations have been reported. FBN1 mutations have been found in 20-80% of patients with MFS depending upon the clinical selection of patients and the mutation detection method used. FBN1 mutations have been reported in a wide range of phenotypes in the Marfan spectrum; neonatal MFS is associated with some FBN1 mutations in exons 24-32; mutations of various types throughout FBN1 have been associated with classical MFS; and FBN1 mutations have also been reported in many of the non-classical Marfan related phenotypes. The Ghent criteria propose that an FBN1 mutation is only of help diagnostically if it has been previously found in a person who independently meets the criteria for diagnosis of MFS. Other than mutations causing neonatal MFS, which occur in exons 24-32, correlation between genotype and phenotype is poor. The majority of patients with cysteine substitutions have classical MFS, and cysteine substitutions in exons 26-32 appear to be associated with classical disease manifesting early in life. Missense mutations that affect the calcium binding site of the cbEGF domain (reducing calcium binding affinity) are associated with varying phenotypes that are likely to be related to the location, calcium binding properties, and domain context of the affected domain within fibrillin-1. There is marked phenotypic variation between subjects with nonsense mutations, which is thought to be due to the degree of nonsense mediated decay of the mutant cDNA. Intrafamilial variation of phenotype is also seen, suggesting that there are factors in addition to the FBN1 genotype that can modify the phenotype. 22 Consequently, knowledge of the underlying FBN1 mutation in an affected proband is of relatively little help either in assigning the appropriate clinical diagnosis or in advising on the likely clinical course or severity of phenotype. Here we report our experience in the Oxford Marfan Clinic, where mutation detection has been performed on 35 families on a research basis using heteroduplex analysis of genomic amplicons with conformation sensitive gel electrophoresis (CSGE) or denaturing high performance liquid chromatography (DHPLC). METHODS The clinical features of the probands are given in table 1. All patients underwent a thorough clinical examination, including slit lamp examination and 2D echocardiogram. Magnetic resonance imaging (MRI) of the lumbosacral spine for evidence of dural ectasia was performed in 14 of the probands. Of the 35 probands, 22 fulfilled the Ghent criteria for MFS (table 1). Two further subjects had two major criteria but involvement of a third system was not demonstrated. The remaining 11 subjects had phenotypes related to MFS. Other family members were also examined for Marfan syndrome where practicable. Mutation detection was performed initially on 17 subjects using heteroduplex analysis of genomic amplicons and CSGE with DNA sequencing, as has been previously described. In subject 12, where a mutation had not been discovered using CSGE, the cDNA was directly sequenced, as a defect had been observed in fibrillin-1 pulse chase studies. DHPLC was used to screen for mutations in the remainder of the patients, including five cases where a mutation had not been found previously using CSGE (cases 2, 5, 7, 15, and 19). Sensitivity of the conditions used for DHPLC were tested by ensuring that DHPLC was able to identify 13 known FBN1 mutations or polymorphisms which had been previously detected by CSGE. The 14 mutation negative subjects only analysed with DHPLC were not rescreened using CSGE, as DHPLC was the more