2009; 15:826-832 <http://www.molvis.org/molvis/v15/a85>
Received 26 March 2009 | Accepted 17 April 2009 | Published 23 April 2009
The first two authors contributed equally to this work.
1Department of Ophthalmology, Harbin Medical University the 2nd Affiliated Hospital, Harbin, China; 2Center of Integrative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China
Correspondence to: Dr. Yanhua Qi, Department of Ophthalmology, Harbin Medical University the 2nd Affiliated Hospital, 246 Xuefu Road, Harbin, Heilongjiang, 150086, China; Phone: 86-451-86605851; FAX: 86-451-86605116; email: firstname.lastname@example.org
Purpose: To identify the molecular defects in the fibrillin-1 gene (FBN1) in two Chinese families with ectopia lentis (EL) and marfanoid habitus.
Methods: Five patients and eight non-carriers in the two families underwent complete physical, ophthalmic, and cardiovascular examinations. Genomic DNA was extracted from leukocytes of venous blood of these individuals in the families as well as 100 healthy normal controls. Polymerase chain reaction (PCR) amplification and direct sequencing of all 65 coding exons of FBN1 were analyzed. The functional consequences of the mutations were analyzed with various genomic resources.
Results: Two novel mutations of FBN1 were identified in our study. One is a splice defect in intron 17 (IVS 17–1G>T) adjacent to exon 18. The other is c.6182G>T in exon 50, which results in the substitution of cysteine by phenylalanine at codon 2,061 (p. C2061F). We provided strong evidences that the splice mutation would potentially lead to the skipping of exons after intron 17 and that the missense mutation at codon 2,061 (p. C2061F) would destroy a disulfide bond.
Conclusions: We detected two novel mutations in FBN1. Our results expand the mutation spectrum of FBN1 and help in the study of the molecular pathogenesis of Marfan syndrome and Marfan-related disorders.
Ectopia lentis (EL; OMIM 129600) is an inherited connective disorder characterized by lens dislocation, often connected with stretched or discontinuous zonular filaments . In most cases, EL occurs as one symptom of Marfan syndrome (MFS; OMIM 154700), a genetic autosomal dominant disorder that is characterized by manifestations mainly involving the cardiovascular, skeletal, and ocular systems . According to the Ghent nosology, a clinical diagnosis of MFS requires the involvement of all three systems with two major diagnostic manifestations . Other disorders such as isolated EL or predominant EL with some skeletal features belong to Marfan-related disorders.
Both Marfan syndrome and Marfan-related disorders mainly result from mutations in the fibrillin-1 gene (FBN1) . FBN1 encodes a 320 kDa glycoprotein consisting of 2,871 amino acids and is located on chromosome 15q21. FBN1 is mainly composed of three types of repeated modules. The first one is the epidermal growth factor (EGF)-like module, which includes six cysteine residues. There are 47 such modules, and most of them are calcium binding (cb) EGF-like modules. The second type is called transforming growth factor β1-binding (or TB) protein-like module (TGF β1-BP-like module, or 8-Cys/TB), which is found seven times in FBN1. This module contains eight cysteine residues that form four disulfide bonds. The last one is a hybrid module, which occurs twice .
In this study, we analyzed two Chinese families with EL and marfanoid habitus and detected two novel heterozygous mutations in FBN1 . In each family, the mutation found cosegregated in the patients and was not observed in any of the healthy family members.
In our study, the patients from two Chinese families with ectopia lentis and marfanoid habitus were from the Heilongjiang province in northeastern China. Two patients and six non-carrier relatives in Family 1, three patients and two non-carrier relatives in Family 2, and 100 healthy normal controls were recruited for this study. The study was approved by the Institutional Review Board of Harbin Medical University (Harbin, China). After obtaining informed consent from all the participants, thorough physical, ophthalmic, and cardiovascular examinations were performed.
Blood specimens (5 ml) were collected in EDTA, and genomic DNA was extracted by the TIANamp Blood DNA Kit (Tiangen Biltech Co. Ltd, Beijing, China).
All coding exons of FBN1 were amplified by polymerase chain reaction (PCR) using a set of 59 pairs of primers. The primers for exons 4, 5, 7, 11, 15, 22, 23, 31, 41, 44, 45, 51, and 52 were from those described by Li and coworkers . The others are listed in Table 1. The PCR products were subsequently purified with a TIANgel Midi Purification Kit (Tiangen Biltech Co. Ltd) and sequenced with an ABI BigDye Terminator Cycle Sequencing kit v3.1 (ABI Applied Biosystems, Foster City, CA).
The potential results of the G→T transversion were estimated using information theory as described in the literature . Briefly, potential splice sites were identified by the splice mutation analysis system based on information theory. Thus, the score of the site containing a mutant nucleotide would be significantly changed compared with that of the wild-type splice site. The analysis had been previously used for the interpretation of other mutations [8,9]. We used walker  visualization maps to present the predicted changes in binding sites.
The protein structure file, 1apj, downloaded from the Protein data bank (PDB) database, demonstrates the solution structure of the transforming growth factor beta binding (TB) protein-like domain 6 of fibrillin (residues 2054–2125) . This structure was displayed with the KiNG viewer to show the missense mutation at codon 2061.
In the two families, all the patients (Figure 1A, Figure 2A) in our study showed similar clinical symptoms (Table 2). Bilateral lens dislocation was discovered in the five patients, and none of them displayed any abnormalities in the cardiovascular system by echocardiogram. However, in Family 2, individual I:2 died of congenital heart disease at the age of 30 years old with big hands according to the description of her daughter (II:2), and her granddaughter (III:1) also died of congenital heart disease only four days after birth. It was not clear whether they had any other abnormalities such as EL because they were deceased several years ago and no related medical records were available. As for the skeletal system, arachnodactyly was present in the five patients.
After direct sequencing of FBN1 in the five patients, a splice defect in intron 17 (IVS 17-1G>T) adjacent to exon 18 (Figure 1B) and a missense mutation involving the substitution of cysteine by phenylalanine in exon 50 (p.C2061F; Figure 2B) were discovered in Family 1 and Family 2, respectively. Neither of the two mutations was detected in the healthy family members (Figure 1C, Figure 2C) or any of the 100 unrelated control subjects.
The IVS 17–1G>T mutation located at a highly conserved splice site of intron 17, which has canonical GT/AG ends (Figure 3A). Information theory analysis revealed that the information contents (Ri) value decreased from 9.2 bits to 0.5 bits by the mutation (Figure 3B). The cysteine residue at position 2,061 was also conserved among mammalian species (Figure 4A). Structure analysis of the transforming growth factor β (TGF-β)-binding protein-like domain revealed that C2061 and C2083 formed one of the four disulfide bonds. (Figure 4B) .
In this study, we described two novel heterozygous mutations in FBN1 (IVS 17–1G>T and p.C2061F). Furthermore, we used various genomic resources to analyze the potential functional consequences of these two mutations.
In Family 1, it was a splice mutation in position 1 of the intron 17-exon 18 boundary in the domain of cb EGF-like number 07. EGF-like domains play a major role in the pathogenesis of fibrillinopathies containing 75% of all the FBN1 mutations registered in the FBN1 Universal Mutation Database (UMD) database. Previously, Rogan et al.  showed that the minimum Ri value for a functional splice site was 2.4 in a study of over 100 splice sites. As for the splice mutation in our study, the Ri value decreased from 9.2 bits to 0.5 bits. The mutation of this base would be expected to disrupt the acceptor site and potentially lead to abnormal mRNA splicing and skipping of exons after intron 17. This also supports the observation that splice mutations often lead to a shortened protein, accounting for about 11%–12% of the gene lesions in FBN1 [5,13]. Interestingly, the c. 2168–1G>T splice site mutation (in IVS 17) involved the same nucleotide of the c. 2168–1G>A substitution previously described in FBN1 .
Family 2 carried a missense mutation affecting cysteine residues in exon 50 in the domain of 8-Cys/TB number 06. This supports the previous studies that mutations involving cysteine substitution are usually associated with EL [13,15,16]. Each 8-Cys/TB module contains eight highly conserved cysteine residues holding TGF-β in an inactive complex in various tissues including the extracellular matrix . Structure analysis showed C2061 and C2083 form one of the four disulfide bonds. Therefore, the substitution of cysteine by phenylalanine in this position was likely to destroy the disulfide bond and cause domain misfolding and structure instability. Recent studies demonstrated that increased TGF-β signaling contributed to selected symptoms of MFS  and could cause dysregulation of cytokine function in mouse models of MFS . All above show that 8-Cys/TB domains also play an important role in the pathogenesis of fibrillinopathies.
Since FBN1 cDNA was cloned and the first mutations of FBN1 were identified in MFS patients in 1991 [20-22], currently more than 1,200 FBN1 mutations have been described . Most of them are missense mutations, and others are nonsense mutations, splice defect, deletions, and so on. In this study, we described two novel heterozygous mutations in FBN1 in the Chinese patients with ectopic lentis and marfanoid habitus and analyzed the potential functional consequences of the two mutations. Our data further expand the mutation spectrum of FBN1 and help in the study of molecular pathogenesis of Marfan syndrome and Marfan-related disorders.
The authors are grateful to all the patients and their families as well as to the normal volunteers for their participation in this study.