Classification and Interpretation for 11 FBN1 Variants Responsible for Marfan Syndrome and Pre-implantation Genetic Testing (PGT) for Two Families Successfully Blocked Transmission of the Pathogenic Mutations

Abstract

Background: The lifespan of Marfan Syndrome (MFS) patients is shortened, especially in patients without early diagnostics, preventive treatment, and elective surgery. Clinically, MFS diagnosis is mainly dependent on phenotypes, but for children, sporadic cases, or suspicious MFS patients, molecular genetic testing, and mainly FBN1 mutation screening, plays a significant role in the diagnosis of MFS. PGT-M gives couples that had a family history of monogenic disorders the opportunity to avoid the occurrence of MFS.

Methods: In this study, 11 families with MFS were recruited and complete clinical features were collected. Variants were classified and interpreted through pedigree analysis according to guidelines. Two families chose to undergo PGT-M; 16 blastocysts were biopsied and amplified. Haplotype analysis was performed to deduce the embryo’s genotype by using single nucleotide polymorphisms (SNPs) identified in each sample.

Results: We identified 11 potential disease-causing FBN1 variants, six of which are novel. All variants were assessed with prediction tools to assess mutation pathogenicity, population databases to evaluate population allele frequency, literature databases to identify whether the variant had been reported in MFS patients, and multiple sequence alignment to carry out conservative analysis. Finally, nine variants were classified as likely pathogenic/pathogenic variants. Among 11 variants, eight variants were missense, and seven of them were located in the Ca-binding EGF-like motifs, moreover, half of them substituted conserved Cysteine residues. We also identified a splice site variant, a frameshift variant, and a synonymous variant. There are two variants that are de novo variants. PGT-M helped two MFS families give birth to a healthy baby not carrying the FBN1 mutation.

Conclusions: In the present study, the FBN1 mutation spectrum was enriched, and may help further elucidate the pathogenesis, benefiting clinical diagnosis and management of MFS. We make use of a reliable PGT-M method for the successful birth of healthy babies to two MFS families.

Introduction

Marfan syndrome (MFS, OMIM 154700) is a connective tissue disease that is caused by mutations in fibrillin-1; it mainly affects several systems including cardiovascular, ocular, and musculoskeletal systems. MFS is inherited in an autosomal dominant pattern with an estimated prevalence of 1–5/10,000 (Ramirez and Dietz, 2007; Radke and Baumgartner, 2014). Clinically, cardiovascular manifestations (aortic dilatation and dissection) are the most serious complications that cause the death of MFS patients, although ocular and skeletal involvement may also pose a great burden on MFS patients (Ammash et al., 2008). Multiple studies confirmed that the lifespan of MFS patients is shortened, especially in patients without diagnostics and surgical treatment (Krause, 2000; Yetman et al., 2003; Groth et al., 2018).

FBN1 gene (OMIM 134797) contained 66 exons and encoded fibrillin-1 protein; its heterozygous mutations are detected in most patients with MFS (Holcomb, 2000). Compound heterozygous or homozygous FBN1 variants in MFS patients are rare (Arnaud et al., 2017). There are 2890 FBN1 mutations that had been described in the professional Human Gene Mutation database. However, more pathogenic genes or atypical mutations in specific populations remain to be identified and interpreted (Arslan-Kirchner et al., 2008). Clinically, MFS diagnosis is mainly dependent on manifestations or phenotypes (Loeys et al., 2010), but for children or suspicious MFS patients, molecular genetic testing, and mainly FBN1 mutation screening, plays a significant role in the diagnosis of MFS (Cañadas et al., 2010; Groth et al., 2017). There is a specific guideline for the interpretation of sequenced variants in the FBN1 Gene for Marfan Syndrome besides the American College of Medical Genetics (ACMG) guideline to help us identify novel mutations in FBN1 likely to cause MFS (Muiño-Mosquera et al., 2018). With the identification of FBN1 as the genetic basis of MFS (Stark et al., 2020), the hope for MFS patients’ early diagnosis, preventive treatment, and elective surgery is feasible.

Pre-implantation genetic testing for monogenic diseases (PGT-M) is a part of the in vitro fertilization (IVF) process, which genetically profiles oocytes or embryos before implantation, and is generally available for any monogenic condition in which the causative variant is known (Besser et al., 2019; Brown, 2020; Doroftei et al., 2020). PGT-M gives couples that had a family history of monogenic disorders the opportunity to avoid the occurrence of such diseases (Hamid and Loyd, 2012; Arian et al., 2020). There are two crucial problems for couples who choose to do IVF with PGT-M: determining the disease-causing variant and effective biopsy and subsequent genetic analysis which prevent any damage to embryo viability (Priner et al., 2019).

In this study, we investigated the clinical manifestations and molecular basis of 11 unrelated suspected MFS families, to screen and identify disease-causing mutations, and two MFS families want to block the transmission of the disease by PGT-M. We reported a successful application of targeted capture sequencing and haplotype analysis-based PGT in MFS families, coupled with prenatal testing for fetal aneuploidy and large chromosomal imbalance arrangement, to help give birth to a healthy baby.

Materials and Methods

Study Subjects

Eleven nonconsanguineous families with MFS were recruited from International Peace Maternity and Child Health Hospital in this study. The ethics committee of International Peace Maternity and Child Health Hospital approved the project and investigators followed the principles of the Declaration of Helsinki. Informed consent was obtained from each patient and their related families before genetic testing. MFS was diagnosed according to Ghent criteria by cardiologists, ophthalmologists, internists, and geneticists.

Clinical Data Collection

Clinical data were retrospectively collected based on patients’ medical records kept at our hospital. Case inclusion criteria and clinical data inclusion scope are (Radke and Baumgartner, 2014): diagnosed patients or family history of MFS (Ramirez and Dietz, 2007); Cardiovascular system phenotype: aortic dilatation, aortic dissection, or mitral valve prolapse (Ammash et al., 2008); Ocular system phenotype: high myopia >6.0D or ectopia lentis (Yetman et al., 2003); Skeletal system phenotype: arachnodactyly, scoliosis, pectus excavatum, or flatfeet.

DNA Extraction and Mutation Detection

Genomic DNA was isolated from peripheral blood or using the MagNA Pure LC DNA Isolation Kit (Roche Diagnostics, GmbH, Mannheim, Germany). Whole-exome sequencing library construction and sequencing were performed using the Illumina platform by Beijing Genomics Institute (BGI) according to the manufacturer’s protocols. The detection covers exons (over 180,000) and 10bp flanking sequences of 22,000 genes. Exome sequencing was performed on the HiSeq2000 sequencing platform (Illumina). In-solution whole-exome capture and massively parallel sequencing was performed using the Agilent SureSelectXT All Exon Kit 51 Mb. Sequenced reads were collected, filtered for quality, and aligned to the human reference sequence (UCSC Genome Browser hg19) with the Burrows-Wheeler Aligner. On average, over 95% of exons were covered at >20×. Sequence variants including single-nucleotide variants (SNVs) and small insertions or deletions (indels) were annotated by ANNOVAR software. Common variants (defined as 10% frequency in 1,000 Genomes) were excluded if they were present in the dbSNP (v.142) database, the 1,000 Genomes Project, or the Exome Aggregation Consortium (ExAC) Browser. The detected variants were annotated and filtered with Annovar based on public databases [such as Mendelian Inheritance in Man (OMIM), Exome Aggregation Consortium (ExAC) Browser and MutationTaster2] in accordance with the criteria set by the American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines. We focus on screen mutations of the FBN1 (NM_000138.5) gene with bioinformatics analysis of FASTQ files. Each mutation we found will be confirmed by bidirectional Sanger sequencing.

Familial Segregation and Classification of Variants

History of 2 Marfan Syndrome Families Who Undergo PGT-M

Proband in family five is a female (III-2). She was a 26-year-old woman who was diagnosed with Marfan syndrome (MFS). Her mother and grandfather are all MFS patients and are treated for to aortic dilatation and mitral valve prolapse. She and her mother are very tall. All three MFS patients in this family suffered cardiac anomalies and skeletal dysplasia. Her father, husband, and aunt were apparently healthy. The variant is classified as uncertain significance with a pathogenic possibility of 67.5%–81.2. The patient had a strong desire to block the disease and to have an unaffected child via PGT-M. After expert consultation, informed consent from the patient and her husband, and approval from the ethics committee, the couples were determined to be pregnant through IVF and PGT-M, and signed an informed consent form for the PGT-M cycle.

Proband in family eight is a Male (I-2). He was a 36-year-old man who was diagnosed with Marfan syndrome (MFS). He had a c.5498G > T [p(Cys1833Phe)] mutation in FBN1 gene. He was a typical MFS patient with disease phenotype in the Cardiovascular system, Ocular system, and Skeletal system. The variant is classified as pathogenic which is a pathogenic possibility over 99.7%. The MFS patient and his wife decided to undergo an IVF cycle associated with PGT-M and signed their informed consent.

Preimplantation Genetic Testing and Prenatal Diagnosis

Family five and family eight obtained 18 and 10 embryos respectively; embryo biopsy was performed on Day 3 (cleavage stage) and blastocysts biopsy was performed in embryos of grade 3 or higher according to Gardner’s grading scale on day 6. The multiple displacement amplification (MDA) products and gDNA libraries were prepared and captured using a 1.5 Mb customized probe covering 350 kb upstream to 350 kb downstream of the FBN1 gene. The SNPs identified in the couple and their parents were used for haplotype construction. Embryos diagnosed as unaffected were selected for transfer. Prenatal molecular diagnosis was performed through amniocentesis at the 13th-20th gestational week. The fetal genotype was confirmed by Sanger sequencing.

Results

Clinical Characteristics of MFS Probands and Patients

Clinical information of MFS probands and patients was summarized in Table 1. Affected patients from these families exhibited similar clinical symptoms of MFS. All the healthy family members had no features of MFS. We recruited 11 probands with MFS and recorded the phenotype of all MFS patients. The phenotype and family history of affected patients with FBN1 mutation coincided with MFS except in Family 4 and Family 6, the clinical manifestations of the two families are mainly ocular defects including ectopia lentis and high myopia >6.0D. The clinical feature of the proband in Family 11 is missing since the proband died before birth.

FBN1 Mutation Screening and Pedigree Analysis

In this study, we analyzed the genomic DNA of 11 probands with MFS. The quality and reliability of targeted NGS data were evaluated based on the percentage of readable bases and the coverage depth in the targeted region, to ensure complete sequencing coverage of all coding regions in candidate genes. Altogether, 11 potentially disease-causal FBN1 gene variants were screened out in 11 probands, 6/11 (54.5%) variants had not been reported before and 5/11 (45.5%) variants had been reported in MFS patients. We showed all mutations and their location in the protein domain of FBN1 (Figure 1). The hot spot mutations were considered to happen frequently in exon 1–16 and secondarily exon 32–40. Among 11 variants, six variants marked in red were first reported through this study. The location and basic information of all mutations are summarized in Table 2. Except for one splicing variant, nine variants are in Ca-binding EGF-like domains and one is not in a Ca-binding EGF-like domain. There are eight missense variants, and four of eight (50%) missense variants affected conserved Cysteine residues of fibrillin 1 protein.

FIGURE 1

Pedigree analyses were performed to obtain familial segregation data and determine whether the mutation is a de novo mutation. Each variant, considered as a causative candidate and pathogenic mutation, was further validated using the Sanger sequencing method in other family members. Figure 2 is the pedigree chart of 11 MFS families. FBN1 mutations in Family 3 and Family 11 were do novo mutations. Figure 3 showed the validation result of FBN1 mutations in MFS families by sanger sequencing.

FIGURE 2

FIGURE 3

FBN1 Variant Classification According to the ACMG Guidelines

To identify disease-causing mutations for MFS families, stringent criteria according to ACMG guidelines were performed. We carefully examined all available literature and mutation-related database for sequence variant interpretation. In Table 3, allele frequencies of variants detected in populations, pieces of computational evidence of pathogenicity prediction, the clinical significance of variants in Clinvar, and whether the variant is a de novo variant are listed. We explored the classification evidence for each variant and did sequence variant interpretation according to the ACMG criterion. In Figure 4, conservation analysis of the related homologous proteins in eight FBN1 missense mutation sites was conducted by referring to the UniProt database. Orthologous protein sequence alignment for eight missense mutations showed the eight mutation sites happened in a highly conserved region of FBN1 among different species. Finally, we confirmed 11 causative candidate and pathogenic heterozygous mutations including five known mutations of the FBN1 gene in the patients, including c.4955G > A [p(Cys1652Tyr)], c.4210+1G > A [p(?)], c.7559C > T [p(Thr2520Met)], c.364C > T [p(Arg122Cys)], and c.3602G > A [p(Cys1201Tyr)], as well as six novel mutations never reported before, including c.4469A > C [p(Glu1490Ala)], c.6615A > G [p(Glu2205 = )], c.3244G > T [p(Gly1082Cys)], c.5885_5895del [p(Tyr1962Serfs*11)], c.5498G > T [p(Cys1833Phe)], and c.6695G > T [p(Cys2232Phe)].

FIGURE 4

For the FBN1 gene, missense variants are a common mechanism of MFS disease (PP2). c.4955G > A [p(Cys1652Tyr)] is a missense variant (PP2), it had been reported by several groups in patients with MFS (PS4_Moderate). The variant is not present in GnomAD and ExAC (PM2). The variant results in a Cysteine substitution in the critical function domain of the calcium-binding EGF-like domain of FBN1 protein (PM1). In Family 1, the mutation was segregated with the phenotype in five affected patients and was absent in unaffected individuals (eLOD = 1.20, PP1_Moderate). Phenotype and family history of this family and reported patients were consistent with FBN1-induced MFS (PP4). Cys1652 is conserved between species and predicted to be disease-causing according to in silico prediction (PP3). In summary, this variation is classified as “likely pathogenic variation”. In August 2016, the patient and his wife became pregnant naturally, and the fetus (II-2) was diagnosed with FBN1 c.4955G > A mutation after prenatal diagnosis.

c.4210+1G > A [p(?)] is in intron 34 and affects the canonical +1 donor splice site most likely leading to abnormal splicing (PVS1). It has been reported previously in association with MFS (PS4_Supporting). The mutation was not detected in the samples of proband’s parent (PM6) and was not present in GnomAD (PM2). In summary, this variation is classified as “pathogenic variation”. In December 2019, the patient and his wife became pregnant naturally, and the fetus (III-1) did not carry this mutation after prenatal diagnosis.

c.7559C > T [p(Thr2520Met)] is a missense variant (PP2), and is found at a very low frequencies in GnomAD (0.00004246) (PM2). Although the variant had been reported in an MFS patient (PS4_Supporting), in our study, the mutation was not segregated with the phenotype in five affected patients and was detected in unaffected individuals (BS4). In summary, this variation is classified as “Uncertain significance variation”.

c.364C > T [p(Arg122Cys)] is a missense variant (PP2) and located in a critical function domain of the not calcium-binding EGF-like domain of FBN1 protein (PM1). The variant is found at a very low frequency in GnomAD (0.000003986) (PM2) and many pieces of prediction software predict this variant to be damaging (PolyPhen2, SIFT, REVEL) (PP3). The variant had been reported in MFS patients before (PS4_Moderate) and was segregated with the phenotype in eight affected patients (eLOD = 2.00, PP1_Strong). In Family 10 and the previous reports, phenotypes and family histories of patients were consistent with MFS (PP4). In summary, this variation is classified as “pathogenic variation”.

c.3602G > A [p(Cys1201Tyr)] is a missense variant (PP2) and results in a Cysteine substitution in the critical function domain of the calcium-binding EGF-like domain of FBN1 protein (PM1). The mutation was not present in GnomAD (PM2) and was predicted to be disease-causing by in silico analysis (PP3). In Family 11, the mutation is a de novo mutation (PM6), and the mutation had been reported in the literature (PS4_Moderate). In summary, this variation is classified as “likely pathogenic variation”. In May 2019, the parents of the probands conceived naturally, and the fetus (II-2) did not carry FBN1 c.3602G > A mutation after prenatal diagnosis.

c.4469A > C [p(Glu1490Ala)] is a novel missense variant (PP2) and located in a critical function domain of the calcium-binding EGF-like domain of FBN1 protein (PM1). The mutation was not present in GnomAD (PM2) and was predicted to be disease-causing by in silico analysis (PP3). In Family 2, the mutation was segregated with the phenotype in four affected patients and was absent in unaffected individuals (eLOD = 0.90, PP1). Moreover, mutation at Glu1490 locus [p(Glu1490Lys)] had been classified as likely pathogenic (PM5). In summary, this variation is classified as “likely pathogenic variation".

c.6615A > G [p(Glu2205 = )] is a novel synonymous variant that had not been reported before, all three MFS patients had c.6615A > G mutation in FBN1 but the other healthy members in her family did not carry this mutation (eLOD = 0.90, PP1). The variant is absent from all population databases (PM2), and multiple lines of computational evidence support a deleterious effect on the gene product through disturb splicing (PP3). The phenotype and family history of the proband in this family are highly consistent with MFS characteristics (PP4). All three MFS patients in this family suffered cardiac anomalies and skeletal dysplasia. In Figure 5, we can see three affected patients have arachnodactyly, flatfeet, pectus excavatum, or pectus carinatum. In summary, this variation is classified as “Uncertain significance variation”. The variant is classified as uncertain significance with a pathogenic possibility of 67.5%–81.2. The patient had a strong desire to block the disease and to have an unaffected child via PGT-M. After expert consultation, informed consent from the patient and her husband, and approval from the ethics committee, the couples were determined to be pregnant through IVF and PGT-M, and signed an informed consent form for the PGT-M cycle.

FIGURE 5

c.3244G > T [p(Gly1082Cys)] is a novel missense variant (PP2) and located in a critical function domain of the calcium-binding EGF-like domain of FBN1 protein (PM1). The mutation was not present in GnomAD (PM2) and was predicted to be disease-causing by in silico analysis (PP3). In Family 6, the mutation was segregated with the phenotype in six affected patients and was absent in unaffected individuals (eLOD = 1.50, PP1_Strong). In summary, this variation is classified as “likely pathogenic variation”.

c.5885_5895del [p(Tyr1962Serfs*11)] is a novel frameshift variant that likely leads to a truncated protein (PVS1). The variant is absent from all population databases (PM2), and the phenotype of affected patients is consistent with MFS (PP4). In summary, this variation is classified as “pathogenic variation”.

c.5498G > T [p(Cys1833Phe)] and c.6695G > T [p(Cys2232Phe)] are all novel missense variants (PP2) and result in a Cysteine substitution in the critical function domain of the calcium-binding EGF-like domain of FBN1 protein (PM1). The mutations were not all present in GnomAD (PM2) and were predicted to be disease-causing by in silico analysis (PP3). In Family eight and Family 9, the phenotype of affected patients is consistent with MFS (PP4). Moreover, mutation at Cys1833 locus [p(Cys1833Arg), p(Cys1833Ser)], and mutation at Cys2232 locus [p(Cys2232Tyr), p(Cys2232Arg)] had been reported to be pathogenic (PM5_Strong). In summary, the two variations are classified as “pathogenic variation".

Haplotype Analysis and PGT-M Cycles

In Family 5, we defined the haplotype linked to c.6615A > G and associated with MFS as F0, and the haplotype linked to wildtype allele as F1. In Family 8, we defined the haplotype linked to c.5498G > T and associated with MFS as M0, while the haplotype linked to wildtype allele was defined as M1. The genotypes of 16 embryos were all successfully determined using the Hidden Markov model approach (Figure 6; Table 4). In Family 5, five embryos were free of maternal FBN1 c.6615A > G variant; embryos 2, 7, and 8 were carriers of c.6615A > G variant. In Family 8, five embryos were free of paternal FBN1 c.5498G > T variant; embryos 1, 6, and 8 were carriers of c.5498G > T variant.

FIGURE 6

Prenatal Diagnosis

Embryo 6 in Family 5 and embryo 2 in Family 8 were selected for transfer and a successful pregnancy was confirmed by human chorionic gonadotropin (hCG) and ultrasound examination. Sanger sequencing results showed that the fetus did not carry mutations. This confirmed the accuracy of PGT-M. The chromosome imbalance anomaly results showed that no copy number variant (CNV) larger than 100 kb was identified in the fetus.

Discussion

In this study, we collected clinical features of all MFS patients in 11 families with the monogenetic disease. We found that the clinical phenotypes of MFS patients in the same family are broadly similar and remain different. It is consistent with the reports in the literatures (Ramirez, 1996; Seo et al., 2018). In Family 1, two children (12 and 15 years old) did not have cardiac phenotypes such as aortic dilatation, aortic dissection, and mitral valve prolapse but adults had; it reminded us that molecular genetic testing for children or atypical MFS patients is important and to warn patients to take medical prophylaxis to prevent cardiovascular pathologies causing the highest mortality in MFS (Comeglio et al., 2007; Franken et al., 2017; Manchola-Linero et al., 2018).

Here, we identified 11 potential disease-causing FBN1 variants in patients with Marfan syndrome, and six of them are novel. Among 11 variants, eight variants were missense, and seven of them were located in the Ca-binding EGF-like motifs, moreover half of them substituted conserved Cysteine residues. According to previous reports, Cysteine residues are highly conserved in fibrillin 1 (Corson et al., 2004), mutations affecting Cysteine residues may disrupt one of the three disulfide bonds and critical functional domain (Schrijver et al., 1999; Faivre et al., 2007). Our results proved that Cysteine substitutions in Ca-binding EGF-like domains of fibrillin 1 may be the most common and critical causal for FBN1 induced MFS.

Besides eight missense variants, we also identified a splice site variant, a frameshift variant, and a synonymous variant. C.6615A > G [p(Glu2205 = )] variant had never been reported before, the affected patient who carries this variant is a typical patient, her mother and grandfather all had typical MFS phenotype (pieces of variant classification evidence including: PM2, PP1, PP3 and PP4). All of them underwent cardiac surgery. The proband and her husband hope to give birth to a healthy baby. Many pieces of prediction software predicted the variant will affect mRNA splicing, however, more functional evidence is needed.

According to statistics, over 25% of MFS patients are sporadic cases, and these patients always have the severe neonatal phenotype (Collod-Béroud and Boileau, 2002; Tan et al., 2017; Weerakkody et al., 2018). In our study, II-1in Family 3 and II-1 in Family 11 are sporadic cases, their parents are normal, they had no family history, and the variants are de novo. Obviously, they had a severe disease phenotype, II-1in Family 3 died when she was 40 years old and II-1 in Family 11 died after birth. The clinical criteria in the revised Ghent nosology are not always suitable for children, particularly those sporadic cases. It further proved that molecular diagnosis of MFS is useful and necessary (Toudjarska et al., 2001; Ades and Group, 2007). Further, making the pathogenesis clear can help patients block the transmission of the pathogenic FBN1 mutation by PGT and produce healthy babies.

Due to phenotypic variability, a high rate of sporadic cases, and lack of a genetic or biochemical test for MFS, the actual incidence of MFS may be considerably higher than the reported 1–5/10,000 (Chiu et al., 2014). And clinical demand for PGT has increased as research and cases on MFS increase. Over the past few decades, PGT always used STR as a genetic marker, but it is time-consuming to select appropriate markers. The capture sequencing and linkage analysis of SNPs located nearby the gene of interest provides a convenient and efficient way for PGT-M experiment design (Brezina et al., 2016). In two families that underwent PGT-M, informative SNPs were distributed from upstream to downstream of the FBN1 gene, ensuring any recombination will be identified. We determined each embryo successfully. Embryo 4 in Family five had a recombination event in the maternal allele (869.56 Kb), but it did not influence the genotype deduction of the FBN1 gene, because the recombination loci were outside the gene region. Of course, we did invasive prenatal diagnosis for two families who underwent PGT-M to avoid misdiagnosis (Chen et al., 2018).

Conclusion

We identified six novel variants and five known variants in the FBN1 gene from 11 Chinese families with MFS. We performed detailed classification and interpretation for 11 variants. In the present study, the FBN1 mutation spectrum was enriched and may help further elucidate the pathogenesis, benefiting clinical diagnosis and management of MFS. We make use of a reliable PGT-M method to enable the successful birth of healthy babies for two MFS families.

Abbreviations

ACMG, American College of Medical Genetics and Genomics; IVF, in vitro fertilization; MDA, multiple displacement amplification; MFS, Marfan syndrome; PGT, Pre-implantation genetic testing; PGT-M, Pre-implantation genetic testing for monogenic diseases

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