A novel FBN1 mutation causes autosomal dominant Marfan syndrome
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- Published online on: September 20, 2017 https://doi.org/10.3892/mmr.2017.7544
- Pages: 7321-7328
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Copyright: © Xiao et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Marfan syndrome (MFS) is an autosomal dominant hereditary disease comprising a disorder of fibrous connective tissue involving the ocular, skeletal and cardiovascular systems (1). According to the Ghent criteria, patients with malfunctions of at least two organ systems could be diagnosed with MFS (2). Aortic root dilatation/dissection and lens dislocation were two cardinal manifestations to establish an unequivocal diagnosis of MFS in patients with positive family history. Due to the large clinical variability of MFS, and several other connective tissue disorders with comparable clinical features, distinguishing MFS from those similar syndromes is still challenging.
Increasing evidence indicates that heredity holds a key role in the development of MFS. It has been reported that MFS generally results from mutations in the human fibrillin-1 (FBN1) gene (3,4). At present, >3,000 mutations have been identified in relation to MFS. Most mutations are specific to a family with MFS, whereas ~10% of FBN1 mutations are shared by different families (5). Located at chromosome 15q-21.1 with 65 exons, the FBN1 gene encodes a secreted 350 kDa glycoprotein (6). Human FBN1 protein shares conserved sequences with other species. FBN1 protein constitutes extracellular microfibrils and controls the stability, as well as the microfibril assembly. Mutations within the FBN1 gene may disrupt microfibril formation, leading to abnormalities of fibrillin and eventually weakening the connective tissue (7).
In the present study, the entire coding region of FBN1 was analyzed, and a novel mutation in exon 14 of FBN1 was identified in all affected members. The newly identified FBN1 mutation in a Chinese family with MFS further emphasizes the important role of FBN1 in the mechanism of MFS development. The present study not only expanded the mutation spectrum of FBN1 resulting in MFS development in a Chinese family, but is also likely to aid understanding of the molecular pathogenesis and clinical diagnosis of FBN1-associated MFS.
Materials and methods
Subjects
A family with MFS was recruited from the Shandong Provincial Hospital Affiliated to Shandong University (Jinan, China) (Fig. 1). This study was conducted in accordance to the tenets of The Declaration of Helsinki and was approved by the Institutional Review Boards of the Hospital of University of Electronic Science and Technology of China and Sichuan Provincial People's Hospital (Chengdu, China), and the Shandong Provincial Hospital Affiliated to Shandong University. A total of 383 ethnically matched, unrelated and normal healthy individuals were recruited from the Hospital of University of Electronic Science and Technology of China & Sichuan Provincial People's Hospital (255 males and 128 females; mean age at recruitment 55.26±8.78 years). These control individuals had no medical history associated with any related diseases. Written informed consent was obtained from all participants prior to the study.
Clinical diagnosis
Two of the family members were diagnosed with MFS according to the revised Ghent criteria (2). Non-consanguineous marriages were found in the family; clinical information of the affected family members is summarized in Table I. All members of this family underwent complete physical, cardiovascular and ophthalmologic examinations. Unrelated healthy individuals also underwent the same examinations.
Mutation screening
Genomic DNA samples were extracted from peripheral blood using a Blood DNA extraction kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The whole coding region of FBN1 (NM_000138.4) was amplified by polymerase chain reaction (PCR) with 35 cycles (30 sec at 95°C for initial denaturation, 30 sec for annealing at different temperatures as shown in Table II, and 30 sec at 72°C for extension), using a GeneAmp® PCR system 9700 (Applied Biosystems; Thermo Scientific Inc.). Sequencing primers of all the exons were designed using Primer 5.0 (Premier Biosoft International, Palo Alto, CA, USA; Table II). Amplified PCR products were purified and sequenced directly (BigDye Terminators Sequencing kit) with an Automated Genetic Analysis system 3130 (both from Applied Biosystems; Thermo Fisher Scientific, Inc.). Comparative amino acid sequence analysis of the human FBN1 protein was performed across different species using HomoloGene (https://www.ncbi.nlm.nih.gov/homologene/?term=FBN1). The potentially damaging effects of the mutation on the structure and function of FBN1 was predicted using SIFT (http://sift.jcvi.org) and PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/).
Results
Clinical findings
The parents and two daughters of a family from Shandong, China, were included in the present study (Fig. 1). Other relatives of this family were not willing to be tested and so additional clinical details were unattainable. Two affected patients (I:1 and II:2) exhibited similar clinical symptoms, including ectopialentis, myopia and strabismus (Fig. 2 and Table I). The left eye of the proband (I:1) underwent refractive lensectomy and vitrectomy combined with silicone oil tamponade after retinal detachment 2 years prior to the current study; following retinal re-attachment, silicone oil was removed 3 months later. The two patients both had the same facial and skeletal features, including arachnodactyly, flat feet and dilation of the aortic root (Fig. 3 and Table I). The proband had pectus carinatum and aortic aneurysm. The patient received Bentall surgery and underwent aortic arch replacement 5 years prior to the current study, as their aortic aneurysm ruptured and formed aortic dissection (Fig. 4). The other two members of the family had no features of MFS.
Mutation screening of FBN1
Direct sequencing of the whole coding region of FBN1 detected a novel missense mutation c.1708 T>G (p.C570G), situated at nucleotide 570 in exon 14 of the coding region (Fig. 5A). This heterozygous mutation was detected in the two affected patients (I:1 and II:2) but was not found in the unaffected mother and daughter (I:2 and II:1) of the family and in the 383 ethnically matched healthy subjects. Therefore, c.1708 T>G (p.C570G) cosegregated to the patients with MFS in this family. Multiple sequencing alignment of human FBN1 protein with various species revealed that the novel mutation occurred within a highly conserved region of the calcium binding epidermal growth factor-like (cbEGF) domain (Fig. 5C). This mutation is a T>G transition, converting cysteine to glycine at amino acid 570 (p.C570G). This amino acid substitution in the FBN1 protein was predicted to be damaging by SIFT and PolyPhen-2.
Discussion
It has been reported that MFS is mainly caused by mutations in the FBN1 gene, which was the first gene identified to cause MFS (8). Of all the identified mutations in the FBN1 gene, 38.6% result in a truncated FBN1 protein and 60.3% represent missense mutations across different ethnic groups (9). FBN1 mutations may cause abnormalities in the formation of microfibrils and fibrillin. As a result, connective tissues weaken (10). A novel FBN1 heterozygous missense mutation, c.1708 T>G (p.C570G) was identified within a Chinese family associated with MFS in the present study.
FBN1 is an important component of microfibrils and is expressed in many human tissues, including in zonules, the cardiovascular system, cartilage, tendon and cornea. The protein serves a role in the formation of zonules and is secreted from ciliary bodies of non-pigmented cells (11). FBN1 protein is composed of repeated modules, including cbEGF and transforming growth factor-1 binding protein-like domains, and is responsible for maintaining microfibers in an ordered arrangement (12,13). The majority of identified missense mutations in FBN1 are localized in cbEGF (14). The mutated monomer of FBN1 could interfere with the polymerization of fibrillin and microfiber aggregation (15). FBN1 mutations within cbEGF modules may disrupt the stability of elastic fibers and render FBN1 susceptible to proteolysis. As a result, the transforming growth factor-β signaling activity that affects extracellular matrix formation may malfunction (4,16).
In the present study, a novel c.1708 T>G (p.C570G) heterozygous missense mutation of the FBN1 gene was reported in a Chinese family with MFS. Three similar missense mutations: c.1709G>A (p.C570Y) (17), c.1709G>C (p.C570S) (18) and c.1709G>C (p.C570R) (19) have been reported in sporadic cases; however, clinical data in these studies were not obtained. In this pedigree, c.1708 T>G (p.C570G) in FBN1 was detected in the two patients with MFS (I:1 and II:2). The proband (I:1) initially came to Shandong Provincial Hospital to see an ophthalmologist and was found to suffer from ectopialentis, myopia and strabismus in both eyes. The proband and the affected daughter (II:2) had similar facial and skeletal features of MFS, including arachnodactyly, flat feet and dilation of aortic root. In addition, pectus carinatum, aortic dissection and retinal detachment were also detected in the proband. These findings suggested that the clinical manifestations of the patient with MFS became more evident with age. This mutation was not included in the Exome Aggregation Consortium dataset; c.1708 T>G (p.C570G) of FBN1 was not detected in the mother (I:2) and another daughter (II:1) of this family, or in the 383 unrelated normal controls during the mutation screening in the present study. This indicated that c.1708 T>G (p.C570G) of FBN1 cosegregated with affected MFS patients and may serve an important role in the pathogenesis of MFS development in this pedigree.
The p.C570G mutation of FBN1 identified in this family with MFS resulted in a substitution of a highly conserved cysteine residue for glycine in a cbEGF domain of FBN1. This mutation is predicted to abolish one disulfide bond and thus affect the sixth conserved cysteine (C6) of the cbEGF domain; disulfide bonds are essential for the correct EGF-like domain structure. SIFT and PolyPhen-2 predictions indicated that this mutation is critical to protein function, supporting a possible pathogenic effect of this mutation. Evidence has revealed that most FBN1 mutations are clustered in exons 24–32, a hot spot region associated with classic and severe forms of MFS (17,20); mutations in exons 12–15 encoding cbEGF-like domains (C3-C6) cause a mild phenotype of MFS with possible late cardiovascular involvement (21). Evidence from the present study consistently indicated that the identified heterozygous mutation, c.1708T>G, is located at exon 14 and that this cysteine substitution detected in the proband resulted in pectus carinatum and aortic dissection. These two factors correlated with increasing age. However, evident symptoms were not detected in the young affected daughter (II:2), even though significant dilation of the aortic root was identified. Nevertheless, further functional analyses are required to confirm the role of FBN1 and its underlying mechanisms in MFS.
In conclusion, a novel heterozygous mutation, c.1708 T>G (p.C570G), in the FBN1 gene was identified in a Chinese family associated with MFS. The results from the present study enrich the spectrum of MFS-associated mutations of FBN1 and may aid presymptomatic molecular diagnosis of undetermined cases of MFS.
Acknowledgements
The present study was supported by grants from the Natural Science Foundation of China [grant nos. 81670853 (B.G) and 81371048 (B.G)], the Department of Science and Technology of Sichuan Province [grant no. 2015HH0031 (B.G.)] and the Health and Family Planning Commission of Sichuan Province of China [grant no. 16ZD028 (B.G.)].
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