Targeted next‐generation sequencing reveals the genetic mechanism of Chinese Marfan syndrome cohort with ocular manifestation

Abstract

Background

Marfan syndrome (MFS) is a hereditary connective tissue disorder involving multiple systems, including ophthalmologic abnormalities. Most cases are due to heterozygous mutations in the fibrillin‐1 gene (FBN1). Other associated genes include LTBP2, MYH11, MYLK, and SLC2A10. There is significant clinical overlap between MFS and other Marfan‐like disorders.

Purpose

To expand the mutation spectrum of FBN1 gene and validate the pathogenicity of Marfan‐related genes in patients with MFS and ocular manifestations.

Methods

We recruited 318 participants (195 cases, 123 controls), including 59 sporadic cases and 88 families. All patients had comprehensive ophthalmic examinations showing ocular features of MFS and met Ghent criteria. Additionally, 754 cases with other eye diseases were recruited. Panel‐based next‐generation sequencing (NGS) screened mutations in 792 genes related to inherited eye diseases.

Results

We detected 181 mutations with an 84.7% detection rate in sporadic cases and 87.5% in familial cases. The overall detection rate was 86.4%, with FBN1 accounting for 74.8%. In cases without FBN1 mutations, 23 mutations from seven Marfan‐related genes were identified, including four pathogenic or likely pathogenic mutations in LTBP2. The 181 mutations included 165 missenses, 10 splicings, three frameshifts, and three nonsenses. FBN1 accounted for 53.0% of mutations. The most prevalent pathogenic mutation was FBN1 c.4096G>A. Additionally, 94 novel mutations were detected, with 13 de novo mutations in 14 families.

Conclusion

We expanded the mutation spectrum of the FBN1 gene and provided evidence for the pathogenicity of other Marfan‐related genes. Variants in LTBP2 may contribute to the ocular manifestations in MFS, underscoring its role in phenotypic diversity.

The study aimed to expand the understanding of Marfan syndrome (MFS) and its genetic basis. It identified 181 mutations in Marfan‐related genes, including 94 novel mutations. This study broadens the mutation spectrum of FBN1 and highlights the significance of other Marfan‐related genes in MFS patients with ocular symptoms.

1. INTRODUCTION

Marfan syndrome (MFS, OMIM: 154700) is a rare hereditary disorder of fibrous connective tissue, exhibiting significant clinical variability and pleiotropism. MFS is characterized by multiple organ system abnormalities, including skeletal, ophthalmologic, and cardiovascular systems (Bitterman & Sponseller, 2017; Pyeritz & McKusick, 1979). Symptoms of MFS include increased height, generalized ligamentous and joint laxity, scoliosis, anterior chest deformity, dislocation or subluxation of crystals, myopia, glaucoma, dural ectasia, tooth deformity, widening or atrophic skin texture, aorta idiopathic dilation, cerebrovascular malformation, and pulmonary blebs (Brown et al., 1975; Newman & Tilley, 1979; Pyeritz & McKusick, 1979; Sponseller et al., 1995; Weir, 1973; Westling et al., 1998). It is suggested that the biochemical basis of MFS is the abnormality of connective tissue proteins or ground substances, including collagen primary structure abnormalities and hyaluronic acid synthesis (Appel et al., 1979; Byers et al., 1981; Pyeritz & McKusick, 1981). This disorder can present as an isolated condition or may coexist with other medical complications, including cardiovascular diseases, ocular lesions, and neurological disorders. MFS typically presents with a family history and is inherited in an autosomal dominant pattern in most cases (Mc, 1955). Previous studies have indicated that patients with MFS exhibit clinical heterogeneity, with the most severely affected patients appearing to be sporadic mutations, while familial patients have milder manifestations (Morse et al., 1990). No gender or ethnicity biases have been reported, although sporadic patients are more likely to have severe and early‐onset symptoms (Buntinx et al., 1991; Verstraeten et al., 2016). Furthermore, the clinical variability and heterogeneity of MFS provide a huge challenge for medical professionals in correctly diagnosing the disorder. Currently, there is no specific treatment for MFS, and it can only be managed through medication and surgery to alleviate symptoms. Most patients succumb to complications such as aortic aneurysm rupture and heart failure at the middle age. Therefore, it is crucial to enhance our understanding of the pathogenesis and genetic mechanisms underlying MFS.

MFS is caused by the mutations of the fibrillin‐1 gene (FBN1, OMIM: 134797) on chromosome 15q21, which is composed of 66 exons and encodes a 350‐kDa important structural protein–fibrillin‐1 (Verstraeten et al., 2016). Fibrillin‐1 is the primary constituent of microfibrils, which are widely distributed throughout connective tissues in the body and assembled into tissue‐specific architectural frameworks, particularly in the cardiovascular and musculoskeletal systems. In addition to a signal peptide, fibrillin‐1 is arranged in three structurally distinct regions, including 47 six‐cysteine epidermal growth factor (EGF)‐like domains, seven transforming growth factor (TGF)‐β‐binding domains, and a cysteine‐rich and proline‐rich domain (Pereira et al., 1993; Ramachandra et al., 2015). The EGF‐like and TGF‐β‐binding domains have a critical role in protein structure stability through covalent bonds of three disulfide bridges. Mutations in the FBN1 gene result in a significant deficiency of fibrillin‐1 and a disorder of microfibril structure. This deficiency of fibrillin‐1 can also lead to an increase in TGF‐b, and excessive signaling and activation of TGF‐b have been identified as the primary cause of MFS (Benke et al., 2013). MFS has been associated with over a thousand individual mutations in the FBN1 gene (Lu et al., 2024), and mutations in specific regions of FBN1 can also result in disorders such as Weill‐Marchesani syndrome or other acromelic dysplasias that exhibit features opposite to MFS (Faivre et al., 2003). As a result, establishing clear genotype–phenotype correlations between FBN1 mutations and MFS has been challenging.

Building on foundational genetic insights, our study further elucidates the mutation spectrum of the FBN1 gene, employing advanced panel‐based next‐generation sequencing (NGS) techniques (Lu et al., 2024). However, these reported mutations cannot entirely account for the pathogenesis of all MFS patients. Although heterozygous mutations in the FBN1 gene are responsible for almost all cases of MFS, there are other Marfan‐like disorders that exhibit similar clinical phenotypes (Verstraeten et al., 2016). These disorders include Loeys‐Dietz syndrome (LDS, OMIM: 609192, 610168, 61486, 615582), Shprintzen‐Goldberg syndrome (SGS, OMIM: 182212), Ehlers‐Danlos syndrome (EDS, OMIM: 130000, 130050, 225400), Stickler syndrome (STL, OMIM: 108300), and contractural arachnodactyly (CCA, OMIM: 121050). Distinguishing these disorders from MFS through clinical diagnosis can be challenging. LDS is a systemic aortic aneurysm syndrome and was first recognized as a separate disease in 2005 (Loeys et al., 2005). Compared with MFS, the cardiovascular manifestations in LDS tend to be more severe and present at an earlier onset age. Some LDS subtypes also exhibit to ocular phenotype similar to MFS and are associated with pathogenic genes such as TGFBR1, TGFBR2, TGFB2, and TGFB3 (Bertoli‐Avella et al., 2015; Lindsay et al., 2012; Loeys et al., 2005; Stheneur et al., 2008). Conversely, SGS is characterized by a Marfanoid habitus and encompasses almost all of the cardiovascular, skeletal, ocular, skin, and craniofacial features observed in MFS. Additionally, SGS can also present with mental retardation and skeletal muscle hypotonia, which are not observed in MFS (Shprintzen & Goldberg, 1982). Evidence indicates that SGS is caused by heterozygous mutations in the SKI gene (Carmignac et al., 2012). Similarly, EDS refers to a group of heritable connective tissue disorders that share the common clinical features of articular hypermobility, tissue fragility, and skin hyperextensibility (Malfait et al., 2017). Some EDS subtypes present with symptoms typical of MFS, including myopia, lens abnormalities, congenital hip dislocation, recurrent joint subluxations, and cardiovascular diseases. Pathogenic genes associated with these subtypes include COL3A1, COL5A1, and PLOD1 (Hautala et al., 1992; Nicholls et al., 1996; Schwarze et al., 1997). Contractual arachnodactyly also shares overlapping features with MFS, including ocular manifestation, and is caused by the mutations of the FBN2 gene. The FBN2 gene is highly structurally similar to the FBN1 gene, with the main difference being that the FBN1 gene contains a proline‐rich domain while the FBN2 gene contains a glycine‐rich domain (Zhang et al., 1994). In addition, some genes such as FLNA, KCNQ1, LTBP2, MED12, MYH11, MYLK, NOTCH1, and SLC2A10 are listed as potentially related to MFS in the ClinVar database. However, limited evidence currently exists to support their association with the disease. Therefore, further studies are needed to gain a better understanding of the genetic mechanism of MFS.

In our study, we extended the mutation spectrum of the FBN1 gene and systematically studied whether mutations in the genes mentioned above affect patients with MFS in cohorts by using panel‐based NGS. None of the 17 sporadic cases or families analyzed in this study exhibited mutations in the FBN1 gene. However, we identified mutations in other Marfan‐related genes, including four pathogenic or likely pathogenic mutations in the LTBP2 gene. This observation indicates that variants in the LTBP2 gene may contribute to the ocular manifestations observed in some patients with MFS, underscoring its potential role in the phenotypic diversity of the condition. We identified a total of 181 mutations, including 94 novel mutations, resulting in an overall mutation detection rate of 86.4%. The most prevalent pathogenic or likely pathogenic mutation is a missense mutation FBN1 c.4096G>A. Furthermore, we identified 13 de novo heterozygous mutations in 14 families with MFS.

2. MATERIALS AND METHODS

2.1. Sample recruitment and ethical statement

A total of 318 participants, 195 cases and 123 controls were recruited, including 59 sporadic cases and 88 families (88 probands and their family members). Our study was conformed to the tenets of the Declaration of Helsinki and approved by the Ethics Committee of the Eye and ENT Hospital of Fudan University. All participants signed the informed consent in our study.

2.2. Clinical symptoms evaluation

All cases underwent a comprehensive ophthalmologic examination at the Eye and ENT Hospital of Fudan University within the past three years, which revealed that every patient had ocular features of MFS. Subsequently, they were diagnosed with MFS according to the Ghent criteria before undergoing gene sequencing. Anterior segment measurements such as the axial length (AL), the flattest keratometry reading (K1), the steepest keratometry reading (K2), the average keratometry value (Km), cylindrical power (Cyl), the axis, and the depth of the anterior chamber (ACD) were evaluated using a biometer (IOL Master 700, Carl Zeiss Meditec, Jena, Germany) with the subject in a seated position. All participants underwent the procedure of phacoemulsification, followed by the insertion of an intraocular lens. During the surgical process, aqueous humor samples ranging from 20 to 100 μL and samples of the anterior lens capsule were obtained. In addition, we collected additional relevant information, including sex, age, age of onset, duration of onset, family history, degree of subjective vision loss, and other clinical symptoms. In our study, the diagnosis of MFS was based on a broader clinical evaluation, particularly following current clinical guidelines and the Ghent diagnostic criteria. Specifically, this included taking into comprehensive consideration the patient's family history, physical examination (including assessments of skeletal, cardiovascular, and ocular systems), and genetic testing results.

2.3. DNA sample collection

Peripheral blood samples were collected from all participants, and genomic DNA samples were extracted using the FlexiGene DNA Kit (Qiagen, Venlo, The Netherlands). The resulting DNA samples were then stored in an EDTA tube at −20 degrees Celsius according to the manufacturer's protocol.

2.4. Panel‐based next‐generation sequencing (NGS)

We performed a panel‐based NGS using the Target_Eye_792_V2 chip, which was designed by BGI‐Shenzhen (Beijing Genomics Institute, Shenzhen, China), to sequence all the samples collected in our study. These 792 genes include those associated with common hereditary eye diseases, covering coding exons, flanking intronic regions, and promoter regions. (Table S1) The DNA fragments from all samples were amplified by polymerase chain reactions (PCR) and sequenced on BGIseq‐2000 platform (Beijing Genomics Institute, Shenzhen, China). The data was analyzed using panel‐based NGS according to the manufacturer's protocols.

2.5. Mutation analysis using bioinformatics tools

The sequencing reads were aligned to the reference human genome (hg38) using the Burrows‐Wheeler Aligner version 0.7.10 (BWA, http://bio‐bwa.sourceforge.net/). The average coverage depth for sequencing data was over 300X, and the target gene coverage was about 99.96%. Mutations were called using Genome Analysis Tool Kit Version 3.3 (GATK, https://software.broadinstitute.org/gatk/). The frequency of all mutations was annotated using some databases, such as 1000 Genomes Project (100G, http://browser.1000genomes.org/), the Single Nucleotide Polymorphism Database (dbSNP, https://www.ncbi.nlm.nih.gov/snp/), Exome Sequencing Project v.6500 (ESP6500, https://evs.gs.washington.edu/EVS/), and Exome Aggregation Consortium (ExAC, http://exac.broadinstitute.org/). The threshold value 0.5% of minor allele frequency (MAF) was used to select mutations in our study, the mutations with MAF > 0.5% were identified as likely benign and were not further analyzed. Then, mutations prioritization was annotated by some databases, like ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), Human Gene Mutation Database (HGMD, http://www.hgmd.cf.ac.uk/ac/index.php), and Online Mendelian Inheritance in Man (OMIM, https://www.omim.org/). In addition, potential pathogenic mutations were predicted by bioinformatics tools: Sorting Intolerant from Tolerant (SIFT, https://sift.bii.a‐star.edu.sg/), Polymorphism Phenotyping v2 (PolyPhen‐2, http://genetics.bwh.harvard.edu/pph2/), Mutation Taster (http://www.mutationtaster.org/), Likelihood Ratio Test Query (LRT, http://www.genetics.wustl.edu/jflab/lrt_query.html) and Functional Analysis through Hidden Markov Models (FATHMM, http://fathmm.biocompute.org.uk/). Mutations were classified into five types: pathogenic, likely pathogenic, uncertain significance, likely benign, or benign according to the American College of Medical Genetics and Genomics (ACMG, https://www.acmg.net/).

2.6. Mutation confirmation

Candidate mutations were reviewed by experienced clinical ophthalmologists and geneticists. Moreover, family phenotype co‐segregation analysis was used to confirm candidate mutations.

2.7. Statistical analysis

We calculated the diagnostic yield for both sporadic cases and cases' families based on the classification of pathogenic, likely pathogenic, and uncertain significance. The novel mutation was defined as one not previously reported in the literature or registered in the ClinVar or Human Gene Mutation databases. A de novo mutation was defined as one not detected in the biological parents of the proband. Statistical analysis was performed using Stata software version 15.1 (StataCorp LP, College Station, Texas).

3. RESULTS

3.1. Clinical information

Our study recruited a total of 318 participants, comprising 195 cases (61.3%) with MFS and 123 controls (38.7%). The cases included 59 sporadic instances and 136 individuals from 88 families, which encompassed 88 probands and their family members. The demographic breakdown revealed 173 males (54.4%) and 145 females (45.6%). The age range of participants spanned from 1 to 84 years, with an average age of 32.8 years, although data for 12 participants were missing. Specifically, the cases had a younger average age of 29.0 years, with data for 4 participants missing, and a significant proportion (161, 82.6%) were under 50 years old. The average duration of MFS among the cases was 10.5 years, indicating that most cases developed the condition in childhood.

All cases underwent comprehensive ophthalmologic examinations, diagnosing them with MFS and identifying symptoms such as ectopia lentis, retinal detachment, iris hypoplasia, and corneal flatness. The basic clinical characteristics of the participants are outlined in Table 1, and their detailed clinical characteristics are provided in Table S2. It is confirmed that every patient met the Ghent diagnostic criteria, but specific clinical information for diagnosing MFS is not included.

TABLE 1.

The basic clinical characteristics of all the samples and 195 cases.

	Number	Average age	Sex (males/females)
All samples	318	32.8	173/145
Cases	195	29.0	111/84
Sporadic cases	59	37.3	34/25
Family cases	136 (88 Proband)	25.4	77/59

To validate novel candidate mutations identified in our study, we recruited an additional 754 participants without MFS. This group included individuals with other ocular conditions such as retinitis pigmentosa (RP), macular degeneration (MD), familial exudative vitreoretinopathy (FEVR), Leber's hereditary optic neuropathy (LHON), and Best disease, along with their normal family members. The ophthalmologic symptoms of these diseases are distinct from those observed in MFS.The data on eye characteristics in patients with MFS reveal nuanced age‐related changes. When comparing individuals from the 0 to 18 age group with those older than 18, there is a marginal reduction in corneal diameters (OD‐D1 from 37.77 to 36.95, OD‐D2 from 38.58 to 37.69, OS‐D1 from 37.44 to 36.54 and OS‐D2 from 38.91 to 37.72). The axial lengths (OD‐L from 25.91 to 26.16, OS‐L from 26.25 to 25.95) exhibit slight variations, suggesting subtle restructuring within the ocular anatomy. Additionally, the corneal endothelial cell density experienced a minor decrease in the right eye (from 3047.28 to 2999.88) but a slight increase in the left (from 3321.69 to 3368.67), showcasing the distinct adaptive responses of different ocular components.The data are provided in Table S3.

3.2. Mutation spectrum of Marfan‐related genes

Our study included 147 probands, comprising 59 sporadic cases and 88 familial cases, achieving a total mutation detection yield of 86.4% (127/147), with detection rates of 84.8% (50/59) in sporadic cases and 87.5% (77/88) in familial cases (Table 2). We detected 181 mutations across these cases, with 96 mutations in the FBN1 gene and the remaining 85 distributed among 13 other Marfan‐related genes (Table S3). The FBN1 gene mutations represented the largest proportion at 53.0% (96/181), followed by LTBP2 (9.4%, 17/181), MYLK (7.8%, 14/181), FBN2 (6.0%, 11/181), FLNA (5.0%, 9/181), COL3A1 (4.4%, 8/181), MYH11 (3.9%, 7/181), COL2A1 (2.8%, 5/181), TGFBR2 (2.2%, 4/181), KCNQ1 (1.7%, 3/181), SLC2A10 (1.7%, 3/181), TGFB2 (1.1%, 2/181), TGFB3 (0.6%, 1/181) and TGFBR1 (0.6%, 1/181) (Figure 1a).

TABLE 2.

The mutation detection yield in all probands, sporadic and families.

The number of mutations	Probands	Sporadic cases	Families
1	50.3%, 74/147	45.8%, 27/59	54.5%, 48/88
2	28.6%, 42/147	132.2%, 19/59	27.3%, 24/88
3	7.5%, 11/147	6.8%, 4/59	5.7%, 5/88
Total mutations detection yield	86.4%, 127/147	84.8%, 50/59	87.5%, 77/88

FIGURE 1.

The basic information of all 181 mutations. (a) Distribution, and frequency of 181 mutations identified in this study. (b) Clinical significance of the 181 mutations.

In total, 165 missense mutations (91.0%, 165/181), 10 splicing mutations (5.6%, 10/181), three frameshift mutations (1.6%, 3/181), and three nonsense mutations (1.6%, 3/181) were identified (Table S4), annotated for pathogenicity according to the ACMG guidelines. This resulted in 16 pathogenic mutations, 38 likely pathogenic mutations (21.0%, 38/181), 112 variants of uncertain significance (VUS) (61.9%, 112/181), and 15 likely benign mutations (8.3%, 15/181) (Figure 1b). The most commonly occurring pathogenic or likely pathogenic mutation was FBN1 c.4096G>A.

Among 17 sporadic cases or families without FBN1 gene mutations (13.4%, 17/127), we found 24 mutations in seven genes related to MFS, including LTBP2 (33.3%, 8/24), MYLK (16.7%, 4/24), COL2A1 (12.5%, 3/24), COL3A1 (12.5%, 3/24), FBN2 (12.5%, 3/24), FLNA (8.3%, 2/24), and TGFB2 (4.2%, 1/24). Four of these mutations were classified as pathogenic or likely pathogenic and were novel: LTBP2c.985C>T, LTBP2 c.4760G>A, LTBP2 c.2428+2T>G, and LTBP2 c.4617_4618delTG. However, mutations were not detected in 9 sporadic cases and 11 families, suggesting the possibility of undetected large deletions or insertions, or involvement of other genes.

The majority of cases were heterozygotes, in line with the autosomal dominant inheritance pattern of Marfan‐related genes. Out of 318 participants, 195 were diagnosed with MFS (59 sporadic cases and 136 cases in families). Among them, 28.7%(56/195) exhibited multiple mutations, 53.3%(104/195) had a single mutation, and the remainder had no detectable mutation.

3.3. Distribution conservation of mutations

As shown in Figure 2a, a total of 96 mutations were widely distributed in 48 exons of FBN1 gene, and no mutation was identified in exons 1, 2, 11, 12, 13, 17, 18, 19, 36, 39, 43, 44, 51, 54, 57, 58, 62, or 64. Mutations in the FBN1 gene were found to be mainly located in exon 16 (6.3%, 6/96), exon 14 (5.2%, 5/96), exon 25 (5.2%, 5/96), and exon 34 (4.2%, 4/96), while the remaining 44 exons had at least one mutation. Figure 2b shows a multiple‐sequence alignment from different species of exon 16, which had the highest number of mutations in FBN1. This alignment revealed high evolutionary conservation. Additionally, two mutations (c.4816+1G>A, c.5788+1G>A) were found to be distributed in the introns of the FBN1 gene, including intron 39 and intron 47. Analysis using Human Splicing Finder (HSF, http://www.umd.be/HSF3/) revealed that these two intronic mutations mainly affected the splice donor or acceptor sites. Both of these splicing site mutations were predicted to be pathogenic by ACMG and were not found in the ClinVar, HGMD, dbSNP, 1000 Genomes (G1000), ESP 6500, or ExAC databases.

FIGURE 2.

(a) The exon distribution of 96 mutations in FBN1 gene. (b) The multiple‐sequence alignment from different species of exon 16. The last line is the amino acid sequence caused by mutations.

The 17 mutations in the LTBP2 gene were distributed across 12 exons and one intron, including exons 1, 3, 4, 8, 12, 14, 16, 17, 32, 33, 35, 36, and intron 14 (c.2428+2T>G). The 14 mutations in the MYLK were detected in 9 exons, including exons 8, 10, 14, 15, 18, 19, 24, 29, and 32. In the FBN2 gene, 11 mutations were detected across 8 exons, with exon 28 having three mutations while the remaining 7 exons (exons 1, 8, 11, 48, 54, 61, and 65) each only had one mutation.

Mutations in the FLNA, COL3A1, KCNQ1, and TGFB2 genes were evenly distributed among their exons (nine for FLNA, eight for COL3A1, three for KCNQ1, and two for TGFB2). Both exon 34 and exon 41 for the MYH11 gene had two mutations, while exon 37 and exon 38 had only one mutation, respectively. Two mutations were found in exon 8 of the COL2A1 gene, while only one mutation was identified in exon 2, exon 23, and exon 38. The four mutations in the TGFBR2 gene were in three exons and one intron (c.170‐2A>G). All three mutations detected in the SLC2A10 gene were in exon 2. A single mutation was identified in both TGFB3 and TGFBR1 in our study. The two intronic mutations in the LTBP2 gene and TGFBR2 gene were also considered as splicing sites and predicted to be pathogenic mutations by HSF, but were not found in any databases. Detailed information on the exon distribution of mutations of these 13 genes is shown in Figure S1.

3.4. Novel mutations of this gene cohort

Among the 181 mutations analyzed, 22 reported mutations (12.2%, 22/181) were identified, 65 mutations (35.9%, 65/181) were previously reported in the Clinvar databases or Human Gene Mutation Database, and 94 mutations (51.9%, 94/181) were novel. Within the novel mutations, missense mutations accounted for the vast majority, representing 90.4% (85/94), followed by splicing mutations (4.3%, 4/94), frameshift mutations (3.2%, 3/94), and nonsense mutations (2.1%, 2/94). These novel mutations were found in 14 genes, including 46 in FBN1 (48.9%, 46/94), 15 in LTBP2 (16.0%, 15/94), 6 in FBN2 (6.4%, 6/94), 6 in MYLK (6.4%, 6/94), 3 in COL2A1 (3.2%, 3/94), 3 in COL3A1 (3.2%, 3/94), 3 in FLNA (3.2%, 3/94), 3 in MYH11 (3.2%, 3/94), 2 in SLC2A10 (2.1%, 2/94), 2 in TGFB2 (2.1%, 2/94), 2 in TGFBR2 (2.1%, 2/94), 1 in KCNQ1 (1.1%, 1/94), 1 in TGFB3 (1.1%, 1/94), and 1 in TGFBR1 (1.1%, 1/94). Out of the 94 novel mutations, 26 were classified as pathogenic or likely pathogenic mutations (27.7%, 26/94), while 68 were of uncertain significance mutations (72.3%, 68/94). The basic information on novel pathogenic or likely pathogenic mutations detected in more than one case is shown in Table 3.

TABLE 3.

The basic information of prevalent novel pathogenic or likely pathogenic mutations.

Gene	Nucleotide change	Amino acid change	Function	Clinical significance	Reference
FBN1	c.3545G>T	p.Cys1182Phe	Missense	Pathogenic	This Study
FBN1	c.4816+1G>A	–	Splicing	Pathogenic	This Study
FBN1	c.7807A>G	p.Asn2603Asp	Missense	Likely Pathogenic	This Study
FBN1	c.4064T>C	p.Ile1355Thr	Missense	Likely Pathogenic	This Study
FBN1	c.4152G>A	p.Met1384Ile	Missense	Likely Pathogenic	This Study
LTBP2	c.2428+2T>G	–	Splicing	Pathogenic	This Study
LTBP2	c.4760G>A	p.Trp1587Ter	Nonsense	Pathogenic	This Study
LTBP2	c.985C>T	p.Gln329Ter	Nonsense	Pathogenic	This Study

These novel mutations were found in 33 sporadic cases (55.9%, 33/59) and 40 families (consisting of 40 probands and their families, totaling 88 cases) (45.5%, 40/88). The FBN1 splicing mutation c.1841T>C was the most prevalent novel mutation, detected in four sporadic cases or families, followed by FBN1 c.5596A>G which appeared three times. Six mutations appeared twice (6.4%, 6/94), while 70 mutations were only detected once (74.5%, 70/94), and 16 mutations (17.0%, 16/94) were not found in any sporadic case or family.Out of the 88 cases involved, these include 33 sporadic cases and 55 cases from 40 families; five (5.7%, 5/88) had three mutations; 34 (38.6%, 34/88) had two mutations; and 49 (55.7%, 49/88) had only one mutation.

3.5. Bioinformatic validation of novel mutations

As mentioned previously, the 318 samples contained 59 sporadic cases and 88 families (consisting of 88 probands and their family members, totaling 136 cases). Therefore, we divided these mutations into two categories to confirm their pathogenesis. In 50 sporadic cases (84.8%, 50/59), a total of 66 heterozygous mutations were detected. Among the identified mutations, 33 (50.0%, 33/66) were novel, while the remaining 50% had been previously reported or could be found in databases. According to ACMG, 6.1% (4/66) mutations were predicted to be pathogenic, 16.7% (11/66) were classified as likely pathogenic, 65.1% (43/66) were uncertain categorized as having significance, and 12.1% (8/66) were deemed likely benign. Among these 15 pathogenic or likely pathogenic mutations, 66.7% (10/15) were novel mutations, while the remaining 33.3% had been previously reported or could be found in databases. The pathogenicity prediction result of 10 novel pathogenic or likely pathogenic mutations using bioinformatics tools is shown in Figure 3a.

FIGURE 3.

(a) The pathogenicity prediction of novel pathogenic or like pathogenic mutations in sporadic cases. The x‐axis showed different mutations and the y‐axis showed different tools. The deeper color is, the more pathogenic mutation is. (b) The pathogenicity prediction of 12 de novo mutations in FBN1 gene. (c) The structural model of protein changes by the homozygous frameshift mutation LTBP2 c.4617_4618delTG.

A total of 128 mutations were detected in 77 families (77 probands, 110 cases in total), accounting for 87.5% (77/88) of the analyzed samples. Among these detected mutations, 64 (50%, 64/128) were novel, while the other 64 had been reported or existed in databases. The pedigrees of families containing mutations consistent with co‐segregation is shown in Figure S2. However, due to incomplete clinical information of family members, co‐segregation validation could not be determined for other mutations.

Although 88 families were initially recruited, both the biological father's and mother's DNA samples were able to be sequenced for annotation in only 64 families. Among these 64 families, 13 heterozygous mutations were considered as de novo mutations in 14 families (21.9%, 14/64), as they were not observed in either biological parent. Of these 13 mutations, almost all (92.3%, 12/13) were missense mutations located in the FBN1 gene, and almost all were classified as pathogenic or likely pathogenic mutations. The pathogenicity prediction result of these de novo mutations of the FBN1 gene using bioinformatics tools is shown in Figure 3b. Furthermore, in F143, only one homozygous mutation in the LTBP2 gene (c.4617_4618delTG) was detected in the proband, and this mutation was also present in both of the proband's biological parents who were unaffected by MFS. The structural model of protein change by this homozygous frameshift mutation LTBP2 c.4617_4618delTG was predicted by SWISS MODEL (https://swissmodel.expasy.org/) and was depicted in Figure 3c.

4. DISCUSSION

MFS is a rare genetic disorder affecting fibrous connective tissue, which can cause severe symptoms in a patient's skeletal, ophthalmic, and cardiovascular systems (Bitterman & Sponseller, 2017; Pyeritz & McKusick, 1979). Previous studies have revealed that almost all cases of MFS are caused by mutations in the FBN1 gene (Lu et al., 2024; Verstraeten et al., 2016). While a few studies have identified pathogenic gene mutations that overlap with MFS in affected individuals (Mizuguchi et al., 2004; Stheneur et al., 2008), and some have even documented evidence of other MFS‐related genes on the Clinvar database, none have explored the pathogenic or genetic mechanisms of these genes in MFS patients, particularly those with ocular manifestations.

Our study used panel‐based NGS to identify mutations in the FBN1 gene and other Marfan‐related genes associated with MFS in patients with ocular manifestations. This study recruited a total of 318 participants, consisting of 195 cases and 123 controls, which included 59 sporadic cases and 88 families (88 probands and their family members). Among the 181 mutations detected, 94 were found to be novel, and 12 were de novo mutations. These mutations, which were unevenly distributed across the FBN1 gene and 13 other genes associated with MFS, were found to have the highest percentage (53.0%, 96/181) in FBN1, which is consistent with earlier research findings that FBN1 is the primary pathogenic gene of MFS. Of these 181 mutations, 165 (91.0%) were missense mutations, 10 (5.6%) were splicing mutations, 3 (1.6%) were frameshift mutations, and 3 (1.6%) were nonsense mutations. Furthermore, the ACMG classified these mutations into four different types: 16 (8.8%) were pathogenic mutations, 38 (21.0%) were likely pathogenic mutations, 112 (61.9%) were of uncertain significance, and 11 (8.6%) were likely benign mutations.

The overall mutation detection yield is 86.4% (127/147), which is higher than that seen in the FBN1 gene alone (74.8%, 110/127). In 17 sporadic cases or families, no mutations were detected in the FBN1 gene (13.4%), but mutations in the other seven Marfan‐related genes were discovered, four of which were classified as pathogenic or likely pathogenic mutations of the LTBP2 gene. Collectively, the findings of this study suggest that the inclusion of additional Marfan‐related genes in the testing panel can increase the detection rate of MFS, variants in LTBP2 may have a potential contributory role in the phenotypic spectrum of MFS, particularly in its ocular manifestations. Future animal experiments will be necessary to validate these assumptions. It should be noted, however, that 9 sporadic cases and 11 families still remain undiagnosed despite our genetic testing panel. One reason for this is that some individuals with MFS may have large deletion or insertion mutations that are not identified through current genetic testing methods. Additionally, there could be other genes related to MFS that have not yet been discovered. As such, further research is necessary to gain a more comprehensive understanding of the genetic mechanisms and pathogenicity of MFS. The most common form of pathogenic or likely pathogenic mutation is a missense mutation, specifically the FBN1 c.4096G>A mutation, which has been identified in three sporadic cases or families. Among 181 mutations identified, 155 of them (85.6%) have been found in at least one sporadic case or family. The other 26 mutations (14.4%, 26/181) have been observed in the normal family members of probands and may not be pathogenic mutations of MFS. ACMG has predicted two of these mutations (KCNQ1 c.520C>T COL3A1 c.1427A>G) as likely pathogenic, but their pathogenicity requires confirmation with additional samples. In addition, 96 mutations (51.9%) have been uncovered, and 26 of these (27.1%) have been forecasted to be pathogenic or likely pathogenic mutations.

Genetic methods and bioinformatics tools were used to confirm these detected mutations. The overall mutation detection rate for families (87.5%) was higher than that for sporadic cases (84.8%), which aligns with previous research suggesting a genetic predisposition to MFS (Verstraeten et al., 2016). In sporadic cases, 66 mutations were identified, including 15 pathogenic or likely pathogenic mutations (22.7%). Of these, 10 (66.7%) were found to be novel mutations. The results of pathogenicity prediction analyses indicate that these mutations are highly pathogenic. In family cases, 128 mutations are identified, including 38 pathogenic or likely pathogenic mutations (29.7%), 16 (42.1%) of which are novel mutations. Almost all cases (with the exception of LTBP2 c.4617_4618delTG) are heterozygotes, indicating that a majority of individuals affected by MFS with ocular manifestations exhibit autosomal dominant inheritance, which corroborates previous studies (Mc, 1955). Besides, 13 de novo mutations are identified in 14 families (21.9%), which is not significantly different from research indicating that approximately 25% of MFS cases are caused by de novo mutations (Verstraeten et al., 2016). Almost all of these mutations are located in FBN1 gene. These findings suggest that the FBN1 gene may be more susceptible to de novo mutations in MFS, and the underlying mechanism warrants further investigation.

5. CONCLUSION

Herein, our study identified a total of 181 mutations in 127 probands, resulting in an overall mutation detection rate of 86.4%. Of these mutations, 94 were novel, including 26 pathogenic or likely pathogenic mutations. Notably, four pathogenic or likely pathogenic mutations were identified in cases without mutations in the FBN1 gene. The most commonly observed pathogenic or likely pathogenic mutation in our study was a missense mutation (FBN1 c.4096G>A). Additionally, 14 MFS families with ocular manifestations were found to exhibit 13 de novo heterozygous mutations. Collectively, our study expands the range of mutations observed in the FBN1 gene and provides additional evidence supporting the pathogenicity of the LTBP2 gene in individuals with MFS and ocular manifestations. Despite the mutations identified in our study, not all patients can be explained by these findings. It is possible that large deletions or insertions in FBN1, as well as mutations in other known Marfan‐related genes, may account for some cases. Moreover, there may be additional genes involved in the development of MFS that have yet to be fully explored. Therefore, our study highlights the importance of further investigation into the genetics, mechanisms, and pathogenicity of FBN1 and other Marfan‐related genes.

AUTHOR CONTRIBUTIONS

J.K.L, Z.S.W., and D.M.H. conceived and designed this study. D.M.H. and Z.W.W recruited patients and performed clinical examinations and interpretation. D.M.H, Z.T.Y, Z.J.L collected the clinical samples and clinical data. D.M.H., X.C, Z.T.Y, Y.X.C., and P.Y.T analyzed the sequencing data. D.M.H wrote and revised the manuscript.

FUNDING INFORMATION

This work was supported by Shenyang Science and Technology Project (RC210438).

CONFLICT OF INTEREST STATEMENT

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

ETHICAL APPROVAL

This study was approved by the Ethics Committee of He University, Shenyang, China.

Supporting information

Supplementary Figure 1.

Supplementary Figure 2.

Supplementary Table 1.

Supplementary Table 2.

Supplementary Table 3.

Supplementary Table 4:

Han, D. , Wang, Z. , Chen, X. , Liu, Z. , Yang, Z. , Chen, Y. , Tian, P. , Li, J. , & Wang, Z. (2024). Targeted next‐generation sequencing reveals the genetic mechanism of Chinese Marfan syndrome cohort with ocular manifestation. Molecular Genetics & Genomic Medicine, 00, e2482. 10.1002/mgg3.2482

DATA AVAILABILITY STATEMENT

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.