Introduction

Marfan syndrome (MFS; MIM #154700) is an autosomal dominant disorder of connective tissue. The disease has an incidence of 1/5,000 with over 25% of sporadic cases. In 1991, the fibrillin-1 gene (FBN1; MIM 134797) was identified as a major disease-causing gene of MFS (Lee et al. 1991). FBN1 on chromosome 15q21.1 codes for fibrillin-1, a main component of extracellular microfibrils. Since the identification of FBN1, over 550 mutations have been identified (Collod-Beroud et al. 2003). Although the mutation detection rate varies from 23.5 to 80%, the overall mutation detection rate is approximately 50% even in patients with classic MFS. FBN1 mutations have been also found in individuals who do not satisfy the diagnostic criteria for MFS such as autosomal dominant ectopia lentis (EL; MIM 129600). Therefore, the wide spectrum of variability of MFS is not wholly explained solely by FBN1 mutations. It is possible that another unknown candidate gene or genes causing or modifying the disease might exist. For example, the fibrillin-2 gene (FBN2; MIM 121050) on human chromosome 5q23–q31 shares a high degree of homology with FBN1 and has been known to cause congenital contractural arachnodactyly (CCA; MIM 121050) (Lee et al. 1991). The fibrillin-3 gene (FBN3) on human chromosome 19p13 also has high homology to other fibrillin family members (Nagase et al. 2001). The FBN3 gene is fragmented into 63 exons, transcribed in a 9 kb mRNA that encodes a 2,809 amino acid protein and has overall homology of greater than 60% with either FBN1 or FBN2, and contains multiple EGF-like domains. Expression of FBN3 is highest in brain tissue.

The spectrum of overlapping disorders presenting with FBN1 mutations like EL define the molecular group of type 1 fibrillinopathies (Collod-Beroud and Boileau 2002). Weill-Marchesani syndrome (WMS; MIM 277600) is also a connective tissue disorder characterized by short stature, brachydactyly, joint stiffness, and eye anomalies including ectopia lentis. Fibrillin immunofluorescence studies of skin biopsies from the patients with WMS show a decrease in immunostainable fibers compared to unaffected controls. Linkage analysis has mapped the autosomal dominant (AD) WMS locus to chromosome 15q21.1 (Wirtz et al. 1996). The FBN1 gene was sequenced in an AD WMS family and in flame deletion of 24 nucleotides was found (Faivre et al. 2003a). These studies suggest that WMS is an allelic condition with MFS and one of a type 1 fibrillinopathies. Interestingly, the autosomal recessive (AR) WMS locus has recently been mapped to chromosome 19p13.3–p13.2, where the FBN3 gene is located (Faivre et al. 2003b), suggesting that FBN3 is a candidate gene for MFS as well as AR WMS. We analyzed allelic variants of the FBN3 gene as well as FBN1 gene using conformational sensitive gel electrophoresis (CSGE) and DNA sequencing in Japanese patients with MFS.

Materials and methods

Genomic DNA extraction and polymerase chain reaction (PCR) amplification

Genomic DNA was collected from 12 patients of ten families containing two couples of parent–child that fulfilled the Ghent nosology revised diagnostic criteria (De Paepe et al. 1996) for the diagnosis of MFS and 50 Japanese volunteers who served as normal controls. Written informed consent for genomic examination was obtained from all participants and the Ethic Committee of Hirosaki University School of Medicine approved this study. Genomic DNA was extracted from peripheral blood leukocytes by the QIA amp DNA blood mini kit (Qiagen, Inc.). PCR primers used to amplify the individual exons of FBN1 were derived from previously reported information (Korkko et al. 2002). PCR primers of FBN3 on Table 1 were originally designed. PCR amplification was performed using 40 ng of genomic DNA, 0.25 mmol/l of forward and reverse primers, 2 mmol/l MgCl2, 0.2 mmol/l dNTPs, and 1 U of Taq DNA polymerase. The PCR conditions consisted of initial denaturation at 95°C for 10 min, followed by 35 cycles at 95°C for 25 s, 58–68°C for 25 s, and 72°C for 35 s, with a final extension at 72°C for 10 min.

Table 1 Primer sequences
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Heteroduplex analysis and sequencing

Heteroduplex analysis was performed using CSGE. CSGE was performed essentially using the same conditions as previously described (Ganguly et al. 1993; Korkko et al. 1998). Samples with abnormal CSGE band patterns were directly sequenced in both directions by means of the ABI PRISM Bigdye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). Forward and reverse primers were the same as those used for amplification.

Population studies

One hundred chromosomes from 50 unrelated controls were tested for the identified mutations by using restriction enzyme digestion or direct sequencing to determine recurrent mutations or polymorphisms, and to confirm their association with the pathologic condition under study.

Results

First, we screened FBN1 allelic variants in MFS patients and identified three novel mutations in four MFS patients containing a parent–child couple. These three mutations consisted of the two nonsense variants and one missense variant shown in Table 2. These variants were completely absent from the genomes of 50 normal control samples. The missense mutation on exon 63 (c. 8121 G>C, p. Cys 2663 Ser) was detected in a parent–child couple. Although we tried to analyze the mutations of other family members, we could not obtain informed consent from them.

Table 2 FBN1 and FBN3 allelic variants identified in 12 MFS patients and 50 normal controls
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Next, we identified ten FBN3 allelic variants in MFS patients without FBN1 mutation using CSGE and sequence analysis. All of ten variants were analyzed by restriction enzyme digestion or direct sequencing and compared to 50 normal control samples. All variants of the FBN3 gene were also detected in normal controls in either homozygous or heterozygous status (Table 2).

Discussion

Although mutations of the FBN1 gene cause MFS, the disease-causing genes remain unknown in about half of MFS patients. In this study, mutations of the FBN1 gene were detected in only four of 12 patients with MFS, even using very sensitive CSGE and sequence analysis. In order to investigate the possibility that a portion of MFS might be caused by mutation of another fibrillin gene, we screened mutations of the FBN3 gene and identified multiple allelic variants. However, a responsible gene mutation for MFS was not detected in these patients. Although the number of the cases included in the present study was small, these results suggest that disease-causing mutations in exon regions of the FBN3 gene are either very rare in MFS or that the FBN3 gene is not a responsible gene for MFS. Functional variant analysis of the FBN3 gene or more widespread screening of FBN3 gene variants might determine a relationship between FBN3 and several connective tissue disorders.