Mutation analysis of the FBN1 gene in a cohort of patients with Marfan Syndrome: A 10-year single center experience

Journal Pre-proofs Mutation analysis of the FBN1 gene in a cohort of patients with Marfan Syndrome: a 10-year single center experience Liliana Mannucci, Serena Luciano, Leila B. Salehi, Laura Gigante, Chiara Conte, Giuliana Longo, Valentina Ferradini, Nunzia Piumelli, Francesco Brancati, Giovanni Ruvolo, Giuseppe Novelli, Federica Sangiuolo PII: DOI: Reference:

S0009-8981(19)32100-X https://doi.org/10.1016/j.cca.2019.10.037 CCA 15898

To appear in:

Clinica Chimica Acta

Received Date: Revised Date: Accepted Date:

31 July 2019 16 October 2019 24 October 2019

Please cite this article as: L. Mannucci, S. Luciano, L.B. Salehi, L. Gigante, C. Conte, G. Longo, V. Ferradini, N. Piumelli, F. Brancati, G. Ruvolo, G. Novelli, F. Sangiuolo, Mutation analysis of the FBN1 gene in a cohort of patients with Marfan Syndrome: a 10-year single center experience, Clinica Chimica Acta (2019), doi: https:// doi.org/10.1016/j.cca.2019.10.037

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Mutation analysis of the FBN1 gene in a cohort of patients with Marfan Syndrome: a 10-year single center experience

Liliana Mannuccia,1,*, Serena Lucianob,1, Leila B. Salehia,c, Laura Gigantea, Chiara Contea, Giuliana Longob, Valentina Ferradinib, Nunzia Piumellib, Francesco Brancatid, Giovanni Ruvoloc,e, Giuseppe Novellia,b,c, Federica Sangiuoloa,b,c a

b

c

Department of Biomedicine and Prevention, University of Rome "Tor Vergata", Rome, Italy

Marfan Syndrome Study Group, Tor Vergata Hospital, Rome, Italy

d

e

Medical Genetics Unit, Tor Vergata Hospital, Rome, Italy

Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy

Department of Experimental Medicine and Surgery, Tor Vergata University, Rome, Italy

*Correspondence to: Liliana Mannucci Medical Genetics Unit Tor Vergata Hospital viale Oxford, 81 00133 Rome, Italy email: [email protected] tel:

1

(+39) 0620900672

These authors contributed equally to this work.

1

Abstract Background Marfan Syndrome (MFS) is a chronic, life-threatening, autosomal dominant connective tissue disorder caused by mutations in the FBN1 gene, coding for fibrillin-1. All organ systems may be affected, but particularly the cardiovascular system, eyes, and skeleton. Mortality generally results from cardiovascular complications, mainly aortic dissection. Currently, the diagnosis of MFS is based on the revised Ghent nosology. Molecular analysis of the FBN1 gene reduces diagnostic uncertainty in patients with suspected MFS or MFS-related disorders (MFS-RD). To date, more than 2700 FBN1 mutations are known. Methods Using Next Generation Sequencing (NGS) followed by Multiplex Ligation-dependent Probe Amplification on NGS-negative samples, we screened FBN1 gene on 124 unrelated patients (101 MFS fulfilling revised Ghent criteria, 20 suspected MFS, 3 MFS-RD) enrolled from 2008 to 2018 at the Multidisciplinary Marfan Clinic, Tor Vergata Hospital, Rome. Results An FBN1 variant was identified in 107/124 (86.3%) patients, including 48 novel variants (46 pathogenic/likely pathogenic, 2 VUS). A pathogenic/likely pathogenic variant was detected in 90/101 (89.1%) MFS patients. Our approach allowed early diagnosis for 10 young patients (age 319 years) with suspected MFS. Conclusions This study broadens the mutation spectrum of FBN1, providing a full update of the molecular basis of MFS in Italy.

Keywords: Marfan syndrome, MFS, FBN1, gene variants, NGS

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1. Introduction Marfan Syndrome (MFS; OMIM# 154700) is an autosomal dominant disorder of connective tissue affecting different organs and systems, most commonly the heart, blood vessels, eyes, and skeleton [1,2]. Incidence of the disease is approximately 1:5,000 individuals, with no ethnic, geographic, or gender preference. Aortic root dilatation and acute aortic dissection represent the main causes of morbidity and mortality in MFS [3], but overall the clinical manifestations can show a wide phenotypic variability in terms of tissue distribution, time of onset and severity. The main observations usually include a combination of dilatation or dissection of the ascending aorta, ectopia lentis, lumbosacral dural ectasia, scoliosis, reduced upper-to-lower segment ratio, chest deformities, and other typical skeletal features [2]. MFS belongs to a large class of heritable connective tissue disorders (HCTD) caused by mutations in genes coding for structural proteins or for components of the transforming growth factor β (TGFE) signaling network [4]. Mutations in the FBN1 gene are mainly the cause of MFS [5]. The FBN1 gene (OMIM# 134797) is located on chromosome 15q21.1 and its coding sequence is divided into 65 exons. It encodes for fibrillin-1, a large glycoprotein that forms microfibrils in the extracellular matrix and is widely distributed in connective tissue throughout the body [6]. Besides its structural role, fibrillin-1 is also involved in the regulation of TGFβ signaling. In fact, FBN1 mutations have been correlated with increased TGFβ bioavailability and activity contributing to the multisystem pathogenesis of MFS [7,8]. Fibrillin-1 has a multidomain structure, with 47 repeated “six-cysteine” epidermal growth factor (EGF)–like motifs, 43 of which have calcium-binding (cb) consensus sequences (cbEGF-like motifs), interspersed with seven “eight-cysteine” or TGFβbinding protein (TGFβ-BP)-like domains [6]. To date, the Human Gene Mutations Database (http://www.hgmd.cf.ac.uk) reports more than 2700 mutations in the FBN1 gene [9], distributed throughout all the gene sequence. Approximately 25% of reported FBN1 mutations are de novo and about two-thirds of all FBN1 mutations are missense, with the majority causing the creation or the replacement of a cysteine residue [10]. 3

The diagnosis of MFS is mainly based on the Ghent Criteria (Ghent-1) established by De Paepe and co-workers in 1996 [11], and requires a major involvement of at least two organ systems. An update of the diagnostic criteria for MFS, called Revised Ghent Nosology (Ghent-2), was released in 2010 [2]. An exhaustive comparison of Ghent-1 and Ghent-2 showed a similar performance in the diagnosis of MFS, even if Ghent-2 proved to be easier to use than Ghent-1 [12]. The main differences between Ghent-1 and Ghent-2 consist in the elimination of minor criteria and the attribution of a greater relevance to the genetic testing of FBN1. Moreover, Ghent-2 provides clear indications for the evaluation of the pathogenicity of newly identified mutations. In particular, a pathogenic potential role for MFS is assigned to those FBN1 mutations creating a premature termination codon (nonsense mutations), destroying a cysteine residue, altering conserved residues in the cbEGF-like domains, deleting or inserting nucleotides inside a coding sequence (in or out of frame), or at last involving or altering a splice site [2]. Furthermore, upon identification of a novel FBN1 variant in an index case, Ghent-2 recommends molecular analysis of other family members, both affected and healthy, in order to verify co-segregation with disease in the family and absence in healthy relatives. The Revised Ghent Nosology also offers a valuable tool for the differential diagnosis of other disorders sharing overlapping phenotypes with MFS, the so-called MFS-related disorders (MFSRD) characterized by skeletal features, a family history of aortic complications, dissection or aneurysms, and eye manifestations. Among these disorders, mutations in FBN1 have been associated with three particular conditions: ectopia lentis syndrome (ELS), MASS phenotype (myopia, mitral valve prolapse, borderline aortic root enlargement, skin and skeletal findings), and mitral valve prolapse syndrome (MVPS). The introduction in clinical practice of Next Generation Sequencing (NGS) has strongly improved the molecular diagnosis of many genetic diseases including MFS, reducing time and costs and ameliorating the clinical management of patients [13]. In this study, we report the molecular analysis of the FBN1 gene performed on a cohort of 124 unrelated patients enrolled over a ten-year 4

period (2008-2018) at the Multidisciplinary Marfan Clinic of Tor Vergata Hospital in Rome. The great majority of these patients had a clinical diagnosis of MFS or MFS-RD, but we also included a small group of patients with suspected MFS (sMFS), who did not fulfill the Ghent criteria for a clinical diagnosis of MFS but showed a strongly suggestive phenotype. Suspected MFS meet a major criterion (mainly cardiac) and have a systemic score of 5-6. Alternatively, they are under the age of 14 and therefore might show later other clinical signs that over time may lead them to meet Ghent criteria. From 2008 to 2013, screening of the FBN1 gene on eligible patients was performed by Sanger sequencing. Forty samples with known genotype were then used to validate a targeted Next Generation Sequencing protocol. From 2013 onwards, the molecular characterization of enrolled patients has been routinely performed by NGS followed by Multiplex Ligation-dependent Probe Amplification (MLPA) in patients negative at NGS analysis.

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2. Materials and methods 2.1. Patients We have investigated 124 unrelated patients referred to the Multidisciplinary Marfan Clinic of the Tor Vergata University Hospital, Rome, Italy (“SIMaRaLcode” 12092003), from 2008 to 2018. Patients recruited from 2008 to 2010 were formerly evaluated using the original Ghent criteria. These patients were then retrospectively evaluated based on Ghent-2 and only patients with a matching diagnosis were included in this study. A total of 121 patients have been diagnosed as MFS or suspected MFS, specifically 101 MFS fulfilling the revised Ghent criteria and 20 suspected MFS not fulfilling the revised Ghent criteria. The remaining three patients had a diagnosis of MFS-RD, specifically two MASS and one ELS. Considering the multisystem phenotype of the disease, the clinical evaluation has to be multidisciplinary. The Multidisciplinary Marfan Clinic of the Tor Vergata University Hospital provides a precise program: during the same day, patients are accompanied by a volunteer and undergo several specialist consultations, including an orthopedic, cardiologic, cardiac surgery, dental, and ophthalmological examination. Cardiac ultrasonography and slit-lamp examination are systematically performed in each subject. Skeletal involvement is assessed by X-ray examination after orthopedic evaluation, as well as Magnetic Resonance (MR) or Computed Tomography (CT) for the assessment of lumbosacral dural ectasia in adults. Children aged ≤ 14 years (n=25) are referred to pediatricians as well. At the end of this pathway, patients receive genetic counseling, during which eligibility for FBN1 genetic testing is assessed based on the fulfillment of the Ghent criteria and on family history. In any case, patients fulfilling clinical Ghent criteria are entered into the regional registry for Marfan disease. Genetic test results are then explained to patients and families by a clinical geneticist during a post-test counseling session. Furthermore, if the diagnosis is confirmed, the patient is immediately informed for the next checkup visit. The Center also takes care of those patients positive for the criteria, but negative for the molecular test or those not fully meeting Ghent criteria (suspected MFS). Written informed consent for DNA storage and use for genetic analysis and research purposes was obtained from all the 6

patients and relatives, as required by the Ethics Committee of Tor Vergata Hospital of Rome and in accordance with the Declaration of Helsinki. 2.2. Genomic DNA extraction Genomic DNA (gDNA) has been isolated from 200 μl whole blood using the EZ1 DNA Blood Kit on an EZ1 Advanced XL automatic extractor (QIAGEN GmbH, Germany), following the manufacturer’s instructions. DNA concentration has been estimated by spectrophotometric method for Sanger sequencing and MLPA and with the Qubit® dsDNA HS Assay Kit (Thermo Fisher Foster City, CA) on a Qubit® 2.0 Fluorometer for NGS analysis. 2.3. Sanger sequencing Sanger sequencing was performed using the BigDye Terminator protocol (Applied Biosystems, Foster City, CA, USA), followed by capillary electrophoresis on an ABI 3130XL system (Applied Biosystems, Foster City, CA, USA). Results were interpreted using the Sequencing Analysis v5.2 software (Applied Biosystems, Foster City, CA, USA). Previously published FBN1 intronic primers were used [14]. 2.4. Next Generation Sequencing (NGS) A custom panel including the coding regions and the exon-intron boundaries of FBN1 (RefSeq NM_000138.4, LRG_778t1, NG_008805.2) has been designed using the Ion AmpliSeq™ Designer software (Thermo Fisher, Foster City, CA). Two different pools of primers were obtained, for a total of 258 amplicons. The calculated coverage of the coding sequence with a minimum depth of coverage of 50x is about 97%, with an exon padding of 15 bp. Regions covered less than 50x were backfilled by Sanger sequencing. Libraries have been generated using 20 ng of gDNA, using the Ion AmpliSeq Library Kit v2.0, following the manufacturer's instructions and successively indexed using the Ion Xpress Barcode Adapter Kit. After dilution of all samples at 100 pM, libraries have been pooled for emulsion PCR on the Ion OneTouch™ 2 instrument, using the Ion PGM™ Template OT2 200 kit, according to the manufacturer’s instructions. The Ion Sphere™ Particles have been enriched using the Ion OneTouch™ Enrichment System and the template sequenced on 7

the Ion Torrent PGM platform using the 316v2 or 318v2 chip. All of these instruments and reagents were supplied by Thermo Fisher (Foster City, CA). Binary Alignment/Map (BAM) and variant call format (VCF) files were generated by plugin, preinstalled in the Torrent Suite. BAM files were analysed on the IGV (Integrative Genomics Viewer, www.broadinstitute.org/igv) [15] and with the Ion Reporter™ software (Thermo Fisher, Foster City, CA). Identified variants have been searched through the following databases: HGMD (http://www.hgmd.cf.ac.uk)

[9],

UMD-FBN1

(http://www.umd.be/FBN1/)

[10],

ClinVar

(http://www.ncbi.nlm.nih.gov/clinvar) [16], ExAC v1.0 (http://exac.broadinstitute.org/) [17], and gnomAD v2.1.1 (http://gnomad.broadinstitute.org/) [17]. According to the criteria established in the Revised Ghent Nosology [2], all novel FBN1 variants being either disruptive (i.e. nonsense, frameshift and splice site variants) or removing/introducing a cysteine residue were considered pathogenic. When accessible, segregation analysis has been considered for evaluation of pathogenicity. Classification of novel variants based on the ACMG/AMP (American College of Medical Genetics and Genomics and the Association for Molecular Pathology) standards and guidelines [18] was performed with the help of the VarSome software (https://varsome.com/) [19]. Variants involving splice sites have also been analysed using the Human Splicing Finder v3.1 tool (http://www.umd.be/HSF/) [20]. Mutations were annotated according to the current HGVS nomenclature [21]. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon 1 in the reference sequence (NM_000138.4). All novel variants identified were deposited in the ClinVar Database, where they will be openly available with accession numbers from SCV000986955 to SCV000987002 at the following link: https://www.ncbi.nlm.nih.gov/clinvar/?term=fbn1[gene]. 2.5. Multiple Ligation-dependent Probe Amplification (MLPA) MLPA was performed using 50 ng of gDNA with the SALSA MLPA P065/P066 Marfan Syndrome Probemixes commercially available from MRC-Holland (Amsterdam, the Netherlands), according to the manufacturer protocol [22]. PCR products were then separated on an ABI 3130XL capillary 8

electrophoresis system (Applied Biosystems, Foster City, CA, USA) and analyzed with the Coffalyser.Net software (MRC-Holland, Amsterdam, the Netherlands). 2.6. Statistical analysis Statistical analysis was performed using MedCalc for Windows, version 13.0 (MedCalc Software, Ostend, Belgium). Continuous variables are expressed as mean values ± standard deviation (SD). The Kolmogorov-Smirnov test was used to assess normality. Difference between groups was analyzed using an unpaired t-test. Independence between two nominal variables was assessed with the Fisher's exact test. The values P<0.05 were considered statistically significant.

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3. Results Sequence analysis of the FBN1 gene has been performed in 124 unrelated patients (median age 36.5, range 3-61 years). All the FBN1 variants identified by Sanger sequencing (from 2008 to 2013) were successively detected also by NGS analysis. Conversely, variants identified directly by NGS analysis (from 2013 to 2018) were always confirmed by Sanger sequencing. No variants were identified in regions with low NGS coverage backfilled with Sanger sequencing. Based on the complete Sanger sequencing results from the first 40 samples, sensitivity and specificity of the NGS test were calculated as 96% and 98%, respectively. Screening of the FBN1 gene with our NGS workflow allowed the identification of 99 different FBN1 variants in 104 of 124 (83.9%) patients. The remaining 20 samples with no detectable FBN1 variant after NGS analysis were further analyzed by MLPA, in order to evaluate the presence of gross deletions or duplications in the FBN1 gene. MLPA identified gross FBN1 gene deletions in three patients. In total, NGS followed by MLPA allowed the molecular characterization of 107 of 124 (86.3%) patients. Among the 102 different mutational events identified, 48 had not been previously reported (absent from the following databases: HGMD, UMD-FBN1, ExAC, gnomAD, and ClinVar) and were not found in 100 in-house control chromosomes. Genotypic and clinical characteristics of the 107 patients in which an FBN1 variant was identified are summarized in Table 1 (patients with novel mutations identified in this study, n=48) and Table 2 (patients with previously published mutations, n=59). Segregation analysis was performed by direct Sanger sequencing on at least one first-degree relative (either affected or healthy) for 35/48 patients with FBN1 novel variants (Table 1), for a total of 92 relatives analyzed, and for 41/59 patients with known FBN1 mutations (Table 2), for a total of 88 relatives analyzed. Based on the ACMG/AMP standards and guidelines, 46 novel variants were classified as pathogenic/likely pathogenic and two variants were of uncertain significance (VUS). The latter were identified in case #25 (MFS) and case #35 (MASS).

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Previously reported FBN1 variants identified in this study were already classified as pathogenic/likely pathogenic, except for the variants identified in case #57 (c.1177A>G; rs983129867) and case #97 (c.6917G>A; rs770443276), which were VUS (in italics in Table 2). MLPA was performed also in samples carrying an FBN1 VUS (n=4) and no gene rearrangements were detected. Overall, in this study a pathogenic/likely pathogenic FBN1 variant was detected in 90 out of 101 patients (89.1%) fulfilling the revised Ghent criteria. Patients not fulfilling the revised Ghent-2 criteria were significantly younger than those who did (16.83±12.90 vs 30.21±13.54, 95% CI 7,24-19,52; P<0.0001). Seventeen patients had no detectable FBN1 mutations after both NGS and MLPA and in 7/17 an FBN1 polymorphism was identified. Clinical and molecular details of these patients are summarized in Table 3. Of all 107 FBN1 variants identified, 52 (49%) were missense, of which 35 involved a cysteine residue, 23 (21%) were nonsense, 14 (13%) caused frameshift, 14 (13%) affected splice sites, 1 (1%) was a small in-frame insertion/deletion, and 3 (3%) were gross deletions. Figure 1 describes the FBN1 variants distribution in our cohort of patients. Among gross deletions, one is a novel deletion encompassing FBN1 exons 17-21 [c.(2113+1_2114-1)_(2671+1_2678-1)del]. The second was previously reported by our group [23] and consists of a large deletion involving the whole FBN1

gene

and

extending

beyond

the

15q21

chromosome

region

(chr15:g.48.066.086_49.259.938del). The last one is a large deletion involving exon 3 of FBN1 [c.(247+1_248-1)_(346+1_347-1)del] and was previously described by another group [24]. Of all novel variants identified, 28 (58%) involve one of the 43 calcium-binding EGF-like modules of fibrillin-1. A schematic representation of fibrillin-1 protein domains and localization of novel variants is shown in Figure 2. As for the genotype/phenotype correlation, ectopia lentis (EL) was observed more frequently in patients with missense variants removing or introducing a cysteine residue (MS-Cys) (25EL/35MS-

11

Cys) compared to patients with mutations creating a premature termination codon (PTC) (13EL/37PTC, including both nonsense and frameshift) (P<0.005).

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4. Discussion In this work we report the results of the molecular analysis of the FBN1 gene in a cohort of 124 probands including 101 patients with a clinical diagnosis of MFS, 3 with MFS-related disorders and 20 showing signs highly suggestive of MFS. Molecular analysis was performed by NGS, followed by MLPA in samples where no potentially causal variant had been identified. Using this approach, we were able to identify an FBN1 variant in 107 patients. Forty-eight variants were newly identified in this study. In patients fulfilling the revised Ghent criteria, we identified a pathogenic/likely pathogenic variant with a detection rate of 89.1% (90/101), in agreement with literature data from other large molecular genetic studies on MFS patients fulfilling Ghent criteria [13,25]. Our data also confirm that fulfillment of the Ghent criteria is a good predictor for the presence of an FBN1 variant. In fact, the robustness of selection criteria is the most important determinant of the outcome of mutational studies. The FBN1 mutational spectrum observed in our study mainly consists of single nucleotide variants localized within the cbEGF-like domains of fibrillin-1 (58%). Almost half (49%) of the identified variants are missense, of which 2/3 involve a cysteine residue. Overall, each class of mutational events is represented in a similar proportion as in reference databases [9,10]. Only four variants were identified in more than one pedigree, further confirming the almost “private” nature of FBN1 mutations [10]. Taken together, these results give a picture of the known wide allelic heterogeneity of FBN1 mutations. This feature highlights the opportunity to take advantage of a high throughput approach of molecular analysis as NGS that in our study allowed us the identification of an FBN1 variant in 104 out of 124 probands, with an overall diagnostic yield of 83.9%. The use of MLPA as an additional test for gene dosage assay on NGS-negative samples turned out to be conclusive only in 3% of cases, in line with literature data and database records [9,13]. Besides the allelic heterogeneity of FBN1 mutations, MFS is also characterized by a wide phenotypic variability. Although clinical manifestations of MFS usually include a combination of 13

dilatation or dissection of the ascending aorta, ectopia lentis, chest deformities, and other typical skeletal features, MFS phenotype can be extremely variable in terms of tissue distribution, time of onset and severity. Intriguingly, clinical presentation may vary also in probands carrying the same FBN1 mutation, even within the same family [26,27]. FBN1 mutations can be clustered into two groups and classified as dominant negative (DN) or leading to haploinsufficiency (HI). DN mutations would lead to interference of abnormal (mutated) fibrillin-1 protein on the normal gene product, thus affecting functions as protein folding or protein–protein interactions and resulting in a disorganized extracellular matrix. On the other side, HI mutations would lead to a reduced amount of normal fibrillin-1 protein. Several studies analyzing large cohorts of MFS probands and relatives have tried to find the key to predict the effect of an FBN1 mutation at the protein (and eventually clinical) level. However, the interpretation of current experimental evidence is still controversial and only a limited number of convincing genotype-phenotype correlations has emerged. One of these is the preferential manifestation of ectopia lentis (EL) in patients with FBN1 variants either disrupting or introducing a cysteine residue (MS-Cys) - classifiable as DN - compared to patients that carry mutations leading to a premature termination codon (PTC) - classifiable as HI [25,28,29]. In agreement with these findings, in our study cohort the prevalence of EL in patients harboring MS-Cys variants was significantly higher than in patients carrying PTC mutations (25EL/35MSCys vs 13EL/37PTC, P<0.005). A novel splice variant has been identified in a four-generation family showing an extreme phenotypic variability. This variant, c.6872-1G>T, is predicted to alter the splice acceptor site of intron 55 of FBN1, most probably leading to a defective FBN1 transcript. We identified this variant in 37 family members (19 males, 18 females) showing markedly different clinical signs in terms of age at onset, aortic root size and progression, and involvement of skin, skeletal, and ocular systems. The most interesting finding regards aortic root replacement. Literature data report that MFS patients undergo total aortic root replacement (TRR) at a mean age of 38.3±1.8 years [30]. In the family here described, six affected members underwent early TRR at a mean age of 20.8±3.7 years 14

(range 16-26). In contrast, two other relatives aged 38 and 41 years respectively, have not yet shown any cardiovascular sign. Evidence of intra-familial variability has also been observed for the previously described nonsense mutation c.6169C>T, p.(Arg2057*), which we detected in a proband and his father presenting with different phenotypes. Indeed, the proband manifested bilateral ectopia lentis, skeletal involvement (scoliosis, pectus carinatum, acetabular protrusion), ascending aortic dissection (replacement surgery previously performed), while his father had a slightly dilated aortic root and only minor skeletal signs. An emerging role for epigenetic factors and/or modifier genes has been reported that might explain the remarkable intrafamilial phenotypic heterogeneity of MFS [31]. As reported, we performed FBN1 mutation screening also in 20 subjects with suspected Marfan syndrome who showed a highly suggestive phenotype but did not fulfill the revised Ghent criteria. Among these, a causal or potentially causal FBN1 variant has been identified in 13 patients. Noteworthy, 10 of these 13 patients were <20 years old. It is well known that clinical manifestations of MFS (e.g. ectopia lentis and thoracic aortic aneurysm) can show a variable onset especially in young patients, and may evolve towards classical Marfan phenotype later on. Literature data show that <25% of individuals with an FBN1 mutation have increased aortic diameter by the age of 17 [32]. Among our 10 young patients with an identified FBN1 mutation, only five presented borderline aortic root measurements at first visit, but all of them developed ectasia of the aortic root (Z>3) over a follow-up window of about five years. Among all our Marfan patients, we observed six young subjects (age range 8-19 years) with earlyonset severe cardiovascular and skeletal manifestations. All these patients carry an FBN1 mutation in exon 27 or exon 28, confirming the reported association of a more severe phenotype with mutations in the so-called “neonatal region” encompassing exons 24-32 [29,33,34]. FBN1 gene screening in our pediatric patients was performed following national and international recommendations on genetic testing on minors [35–37]. Children were included in the informed assent/consent process, to the extent that they were capable. In the case of very young patients, 15

genetic counselors illustrated the possibility to defer FBN1 screening until the children are able to participate in their own health care decision making. In accordance with local laws and regulations, the final decision about genetic testing on eligible minors was taken by the parents (or legal guardians). In our 10-year experience, all families decided to have their children tested for FBN1 mutations immediately after the first visit. This included some very young patients (aged 3-10 years) enrolled as suspected Marfan because of the presence of ectopia lentis. A pathogenic/likely pathogenic FBN1 variant was detected in all of these children. Three of these variants were novel, while one had been previously associated with aortic aneurysm [10]. In this last case, genetic testing allowed the identification of a child at risk for aortic complications in only a few months after enrollment. In general, for children with suspected MFS a systematic, long-term clinical follow-up may be necessary to reach a conclusive diagnosis on clinical grounds alone. In some cases, molecular analysis of the FBN1 gene may help early diagnosis, thus offering timely medical benefit through prompt initiation of treatment and/or prevention of aortic dilation, and early adoption of lifestyle adjustments to minimize the risk of aortic damage. No FBN1 mutations were identified in 17/124 patients, specifically 10 MFS, 6 sMFS and 1 EL. This is in line with the reported observation that approximately 10% of MFS patients remain uncharacterized at the molecular level [13]. In our study, it could be partially due to the experimental limitation of our NGS custom design, which does not include deep intronic or regulatory regions of the FBN1 gene. Deep intronic pathogenic variants, most commonly leading to aberrant pre-mRNA splicing, have been described for a number of human genetic disorders [38], but their identification is a rare event if compared to mutations in the coding regions or at canonical splice sites. Nevertheless, such events might be underestimated since standard mutation analysis is usually limited to coding sequences and exon–intron boundaries. To date, a limited number of FBN1 deep intronic mutations disrupting normal splicing have been identified [38–40], and only

16

0.2% of all known FBN1 pathogenic variants have been reported in regulatory regions [9]. In addition, genetic heterogeneity for MFS could not definitely be ruled out. Since several heritable connective tissue disorders can show some degree of clinical overlap with MFS, a differential diagnosis could eventually be considered for our patients with a clinical diagnosis of MFS or suspected MFS who had negative results from FBN1 genetic testing. Most of these patients presented with aortic aneurysm or dissection and 10 of them received a clinical diagnosis of Marfan based on the fulfillment of the revised Ghent criteria (presence of aortic root enlargement/dissection together with a sufficient systemic score and/or ectopia lentis). Among heritable conditions characterized by aortic aneurysms, the differential diagnosis of MFS typically includes Loeys-Dietz syndrome (LDS). The phenotypic overlap between MFS and LDS can include cardiovascular, skeletal, and cutaneous features, and clinical manifestations of LDS can also meet the revised Ghent criteria. Compared to MFS, LDS patients typically show more severe cardiovascular features as widespread aortic and arterial tortuosity and aneurysms. Discriminating features of LDS include hypertelorism, bifid uvula or cleft palate, and clubfoot, while ectopia lentis is not observed in LDS patients [2,4]. LDS is caused by heterozygous mutations in TGBR1/2, TGFB2/3, or SMAD2/3, all coding for components of the TGFβ signaling pathway [2,4,41]. Patients with mutations in TGFBR1/2 or SMAD3 generally show a more severe aortic and arterial phenotype [4]. Patients harboring a SMAD3 mutation usually present osteoarthritis [41]. For patients with MFS or suspected MFS who were negative at FBN1 screening, we could consider extending the molecular analysis to genes associated with LDS and other heritable thoracic aortic diseases, based on the findings from their regular clinical and instrumental re-evaluation. The overall detection rate for FBN1 variants in the study group was 86.3% (107 out of 124 patients). This confirms the key role of a Multidisciplinary Clinic fully dedicated to Marfan Syndrome evaluation, where a periodic follow-up is planned for all patients. During this study, follow-up was scheduled on average every six months (every 3, 6, or 12 months, depending on cardiac surgery check-up) for all patients positive to clinical and/or molecular diagnosis including 17

13 pediatric subjects who did not meet Ghent criteria at first visit. Eight of these patients have revealed other phenotypic manifestations of Marfan syndrome over the years. The remaining five patients have not yet shown other clinical manifestations, although three of them carry a mutation in FBN1. These young patients will be periodically re-evaluated over time. Taken together, these results highlight the importance of an integrated and multidisciplinary clinical evaluation and life-long follow-up of patients diagnosed or suspected of MFS, considering the agerelated expression of skeletal and cardiovascular manifestations [32,42,43]. In conclusion, we confirm that the use of NGS followed by MLPA is a robust, rapid, and costeffective strategy in a daily clinical diagnostic setting. This work broadens the mutation spectrum of FBN1 in the Italian population, thus helping the molecular diagnosis of MFS and, consequently, the clinical management especially in young, pre-symptomatic patients, offering a clear advantage in terms of improved clinical outcomes.

Author contributions Study design: Federica Sangiuolo, Francesco Brancati, Liliana Mannucci, Leila B. Salehi. Data acquisition: Serena Luciano, Liliana Mannucci, Laura Gigante, Chiara Conte, Giuliana Longo, Valentina Ferradini, Nunzia Piumelli, Giovanni Ruvolo. Data analysis and interpretation: Liliana Mannucci, Serena Luciano, Laura Gigante, Valentina Ferradini, Giuliana Longo. Drafting of manuscript: Liliana Mannucci, Serena Luciano, Francesco Brancati, Giuseppe Novelli, Federica Sangiuolo. Critical revision of the manuscript: all authors. Final approval of the version to be submitted: all authors. Declarations of interest None. Acknowledgements 18

We would like to express our sincere gratitude to the patients and their families and to patient associations - especially the “Vittorio Association” - always contributing with their precious work. We are particularly grateful to all the Volunteers of Tor Vergata Hospital for their valuable help and to the members of the Marfan Syndrome Study Group of Tor Vergata Hospital*. The authors would like to thank the Genome Aggregation Database (gnomAD) and the groups that provided exome and genome variant data to this resource. A full list of contributing groups can be found at https://gnomad.broadinstitute.org/about. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

*Marfan Syndrome Study Group of Tor Vergata Hospital: G. Ruvolo (Coordinator); F. Bertoldo, C. Donzelli (Cardiovascular surgery); P. Polisca (Cardiology); G. Novelli, F.C. Sangiuolo, L. B. Salehi (Medical Genetics); R. Mancino, E. Di Carlo (Ophthalmology); P. Bollero (Dentistry); P. Cozza, G. Laganà (Pediatric Dentistry); P. Farsetti, F. De Maio, V. De Luna, F. Mancini (Orthopedics); L. Chini, S. Graziani (Pediatrics); R. Floris, M. Chiocchi, M. Sperandio (Radiology); A. Infante (Counseling); A. De Stefano (Volunteer Association).

19

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26

FIGURE LEGENDS FIGURE 1

FBN1 variants distribution in our cohort of patients. MS-Cys: missense

introducing/eliminating a cysteine residue; indels: insertions/deletions

FIGURE 2

Schematic representation of fibrillin-1 and localization of novel variants identified in

this study. The FBN1 gene encodes for the 2,871 aminoacid multidomain protein fibrillin-1, which contains 47 tandem domains with homology to a module found in human epidermal growth factor (EGF) precursors. Variants affecting splice sites and gross deletions are not reported. cb EGF: calcium-binding epidermal growth factor; EGF: epidermal growth factor; LTBP: latent TGFβ binding protein; TGFBP: transforming growth factor β binding protein; FibuCTDIII: Fibuline Cterminal domain III

27

28

2

2

3

4

5

5

6

8

11

13

13

14

15

15

16

16

#2

#3

#4

#5

#6

#7

#8

#9

#10

#11

#12

#13

#14

#15

#16

#17

c.2053T>C

c.1962_1981delinsAG

c.1888A>C

c.1881_1883inv

c.1782delT

c.1646_1649delinsGT

c.1634G>C

c.1378T>A

c.979A>G

c.634A>C

c.510delC

c.503G>T

c.376G>T

c.298T>C

c.202T>C

c.189T>G

c.176delG

Nucleotide change

p.?

p.Cys685Arg

p.Asp654_Cys661delinsGluGly

p.Asn630His

p.Cys628Asn

p.Phe594Leufs*31

p.Thr549Serfs*9

p.Arg545Pro

p.Cys460Ser

p.Arg327Gly

p.Thr212Pro

p.Tyr170*

p.Cys168Phe

p.Gly126*

p.Cys100Arg

p.Cys68Arg

p.Tyr63*

p.Cys59Leufs*49

Protein change

SPL

MS-Cys

InDel

MS

MS-Cys

FS

FS

MS

MS-Cys

MS

MS

FS

MS-Cys

NS

MS-Cys

MS-Cys

NS

FS

Type of mutation

n.p. n.p. + n.p. + + n.p. n.p. n.p. + + + n.p. +

4-Cys motif LTBP- like 4-Cys motif LTBP- like EGF-like# 01 EGF-like #02 EGF-like #03 EGF-like #03 Hybrid motif #01 cb EGF-like #02 cb EGF-like #04 cb EGF-like #04 cb EGF-like #04 cb EGF-like #05 cb EGF-like #06 cb EGF-like #06

TGFBP #02

TGFBP #02

n.p.

+

+

+

4-Cys motif LTBP- like

TGFBP #02

Segregation studies

Protein domain

Novel FBN1 variants identified in the study cohort

IVS16 c.2113+1G>C

2

#1

#18

Exon

Case

TABLE 1

+

+

-

+

+

+

+

-

-

+

+

+

+

+

+

+

+

+

Ghent criteria (at visit)

MFS

MFS

sMFS

MFS

MFS

MFS

MFS

sMFS

sMFS

MFS

MFS

MFS

MFS

MFS

MFS

MFS

MFS

MFS

Clinical Diagnosis (pre-test)

-

-

-

+

+

+

+

-

-

+

+

+

+

+

-

+

+

+

Systemic score

+

+

-

+

+

+

+

-

-

+

+

+

+

+

+

+

+

+

Cardiac

EL

EL

EL

EL

EL

EL

EL

EL

EL

EL

EL

EL

EL

-

EL

EL

M

/

Ocular

/

/

/

/

/

/

/

/

/

+

/

/

/

+

/

/

/

/

Nervous

-

-

-

-

-

-

-

-

-

+

-

-

-

-

-

-

-

-

Pulmonary

34

45

5

53

44

24

60

10

3

30

18

38

15

37

60

18

38

8

Age at genetic counseling (years)

Pathogenic

Likely pathogenic

Likely pathogenic

Likely pathogenic

Likely pathogenic

Pathogenic

Pathogenic

Likely pathogenic

Likely pathogenic

Likely pathogenic

Likely pathogenic

Likely pathogenic

Likely pathogenic

Pathogenic

Likely pathogenic

Likely pathogenic

Pathogenic

Pathogenic

29

ACMG/AMP classification

24

24

25

27

27

40

41

43

44

46

49

49

#24

#25

#26

#27

#28

#29

#30

#31

#32

#33

#34

#35

c.6145A>G

c.6118T>C

c.5678A>G

c.5498G>A

c.5416T>G

c.5093delA

c.5057delA

c.3398_3399delAG

c.3372_3376delCCGAG

c.3096C>A

c.3065G>A

c.2886C>A

c.2638G>T

c.2561G>A

52

52

54

54

#37

#38

#39

#40

c.6701dupT

c.6634C>T

c.6496+1delG

c.6386A>G

IVS51 c.6380-1G>T

21

#23

#36

21

IVS20 c.2539+2T>G

#21

#22

IVS19 c.2419+2T>G

#20

c.(2113+1_21141)_(2677+1_2678-1)del

17-21

#19

p.Tyr2236Ilefs*8

p.Gln2212*

p.Asp2166Ilefs*19

p.Asp2129Gly

p.?

p.Ser2049Gly

p.Cys2040Arg

p.Asn1893Ser

p.Cys1833Tyr

p.Cys1806Gly

p.Asn1698Thrfs*17

p.Asn1686Ilefs*5

p.Glu1133Glyfs*6

p.Cys1124Trpfs*6

p.Cys1032*

p.Gly1022Glu

p.Tyr962*

p.Gly880Cys

p.Trp854*

p.?

p.?

p.?

FS

NS

FS

MS

SPL

MS

MS-Cys

MS

MS-Cys

MS-Cys

FS

FS

FS

FS

NS

MS

NS

MS-Cys

NS

SPL

SPL

GD

n.p. n.p.

Hybrid motif #02 Hybrid motif #02

+ + + + + + + + +

cb EGF-like #26 cb EGF-like #28 cb EGF-like #31 cb EGF-like #31 cb EGF-like #32 cb EGF-like #32 cb EGF-like #33 cb EGF-like #34 cb EGF-like #34

+

cb EGF-like #24

n.p.

+

cb EGF-like #13

cb EGF-like #25

+

cb EGF-like #13

+

+

cb EGF-like #11

TGFBP #05

+

n.a.

n.p.

+

Hybrid motif #02

TGFBP#03

+

+

n.a.

n.a.

+

+

+

+

+

-

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

MFS

MFS

MFS

MFS

MFS

MASS

MFS

MFS

MFS

MFS

MFS

MFS

MFS

MFS

MFS

MFS

MFS

MFS

MFS

MFS

MFS

MFS

+

+

+

+

+

+

+

+

+

+

+

+

+

-

+

+

+

+

+

+

+

+

+

+

+

+

+

-

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

-

-

EL

EL

M

-

EL

M

-

EL

M

-

EL

EL

EL

EL

-

EL

-

EL

EL

EL

/

/

/

/

/

/

+

/

/

+

/

/

/

/

/

/

+

/

/

/

/

+

-

-

-

-

-

-

+

-

-

+

-

-

-

-

-

-

-

-

-

-

37

53

10

48

39

19

25

31

46

29

40

21

37

10

27

48

30

35

36

45

37

37

Pathogenic

Pathogenic

Pathogenic

Likely pathogenic

Pathogenic

VUS

Likely pathogenic

Likely pathogenic

Likely pathogenic

Likely pathogenic

Pathogenic

Pathogenic

Pathogenic

Pathogenic

Pathogenic

VUS

Pathogenic

Likely pathogenic

Pathogenic

Pathogenic

Pathogenic

Likely pathogenic

30

c.6828T>G

c.6804delC

57

60

63

#46

#47

#48

c.7951G>T

c.7559delC

c.7099G>T

c.6885T>G

p.Glu2651*

p.Thr2520Serfs*162

p.Gly2367*

p.Cys2295Trp

p.?

p.Cys2276Trp

p.Ile2269Leufs*22

p.?

NS

FS

NS

MS-Cys

SPL

MS-Cys

FS

SPL

+

cb EGF-like #43

+

cb EGF-like #36

+

+

cb EGF-like #36

cb EGF-like #39

n.p.

cb EGF-like #35

+

+

cb EGF-like #35

TGFBP #07

+

cb EGF-like #35

+

+

+

+

+

+

+

+

MFS

MFS

MFS

MFS

MFS

MFS

MFS

MFS

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

-

M

M

M

EL

M

EL

-

/

+

/

/

/

/

/

/

-

+

-

-

-

-

-

-

39

30

17

50

18

22

31

43

Pathogenic

Pathogenic

Pathogenic

Likely pathogenic

Pathogenic

Likely pathogenic

Pathogenic

Pathogenic

deletion 31

missense; MS-Cys: missense introducing/eliminating a Cysteine residue; NS: nonsense; FS: frameshift; SPL: splice site; InDel: insertion/deletion; GD: gross

ectasia; (/) indicates unavailable data. Pulmonary involvement: (+) indicates the presence of spontaneous pneumothorax; (-) indicates the feature is absent. MS:

indicates the feature is absent. Ocular involvement includes ectopia lentis (EL) and myopia (M). Nervous involvement: (+) indicates the presence of dural

involvement: (+) indicates aortic root enlargement (Z-score ≥2.0 for subjects aged 20 years and above, Z-score ≥3.0 for subjects younger than 20 years; (-)

fulfillment and (-) indicates non-fulfillment. Systemic score: (+) indicates systemic involvement (score ≥7) and (-) no systemic involvement (score <7). Cardiac

family members and/or absence in healthy family members; n.p.=not performed. Clinical signs of patients are also reported. Ghent criteria: (+) indicates

NG_008805.2. Coding exons are numbered from 1 to 65. Segregation studies: (+) indicates the presence of the same variant in the proband and other affected

(VUS). In one patient diagnosed with MASS phenotype (case #35) a VUS was identified. FBN1 reference sequences are: NM_000138.4, LRG_778t1,

(sMFS) were predicted as pathogenic (n=25) or likely pathogenic (n=21), except than in 1 single case (#25), classified as variant of unknown significance

Forty-eight private FBN1 variants were identified in the present study. Variants identified in patients with a clinical diagnosis of MFS or suspected MFS

56

#45

IVS55 c.6872-1G>T

55

#43

#44

55

IVS54 c.6740-1G>T

#42

#41

Exon

1_65

3

3

3

4

6

6

6

10

IVS11

12

12

13

13

13

18

21

22

Case

#49

#50

#51

#52

#53

#54

#55

#56

#57

#58

#59

#60

#61

#62

#63

#64

#65

#66

TABLE 2

c.2696G>A

c.2638G>A

c.2168A>G

c.1693C>T

c.1670G>A

c.1601G>A

c.1585C>T

c.1585C>T

c.1468+5G>A

c.1177A>G

c.650G>A

c.640G>A

c.586C>T

c.364C>T

c.275G>A

c.266G>A

c.(247+1_248-1)_(346+1_347-1)del

c.(?_-1)_(*1_?)del (chr15:g.48.066.086_49.259.938del)

Nucleotide change

p.Gly899Glu

p.Gly880Ser

p.Asp723Gly

p.Arg565*

p.Cys557Tyr

p.Cys534Tyr

p.Arg529*

p.Arg529*

p.?

p.Met393Val

p.Trp217*

p.Gly214Ser

p.Gln196*

p.Arg122Cys

p.Gly92Glu

p.Cys89Tyr

p.?

p.?

Protein change

MS

MS

MS

NS

MS-Cys

MS-Cys

NS

NS

SPL

MS

NS

MS

NS

MS-Cys

MS

MS-Cys

GD

GD

Type of mutation

Previously published FBN1 variants identified in the study cohort

+

n.p.

+

+

+

+

+

+

n.p.

+

n.p.

+

+

+

+

+

+

+

Segregation studies

+

+

-

+

+

-

-

+

+

-

+

+

+

+

+

-

+

+

Ghent criteria (at visit)

MFS

MFS

sMFS

MFS

MFS

sMFS

sMFS

MFS

MFS

MASS

MFS

MFS

MFS

MFS

MFS

sMFS

MFS

MFS

Clinical Diagnosis (pre-test)

+

+

-

+

+

+

+

+

+

+

+

-

+

+

+

-

+

+

+

+

-

+

+

-

-

+

+

-

+

+

+

+

+

-

+

+

Systemic Cardiac score

EL

EL

EL

-

EL

EL

-

EL

M

-

EL

EL

-

EL

EL

EL

M

EL

Ocular

/

/

/

/

/

/

/

/

/

/

/

/

/

/

/

/

/

/

-

-

-

-

-

-

-

-

+

-

-

-

-

-

-

-

-

-

Nervous Pulmonary

61

24

36

41

25

11

4

25

41

19

45

23

14

16

30

23

40

22

Age at genetic counseling (years)

32

24

24

26

27

27

27

27

27

27

IVS27

28

34

36

37

IVS39

40

43

43

IVS43

44

IVS46

IVS46

#67

#68

#69

#70

#71

#72

#73

#74

#75

#76

#77

#78

#79

#80

#81

#82

#83

#84

#85

#86

#87

#88

c.5788+5G>A

c.5788+5G>A

c.5479A>C

c.5422+1G>A

c.5379T>G

c.5344T>C

c.5021G>A

c.4942+1G>A

c.4588C>T

c.4567C>T

c.4222T>C

c.3497G>A

c.3463+1G>A

c.3408C>G

c.3408C>G

c.3389A>C

c.3386G>A

c.3373C>T

c.3373C>T

c.3302A>G

c.2860C>T

c.2741G>A

p.?

p.?

p.Thr1827Pro

p.?

p.Cys1793Trp

p.Cys1782Arg

p.Cys1674Tyr

p.?

p.Arg1530Cys

p.Arg1523*

p.Cys1408Arg

p.Cys1166Tyr

p.?

p.Tyr1136*

p.Tyr1136*

p.His1130Pro

p.Cys1129Tyr

p.Arg1125*

p.Arg1125*

p.Tyr1101Cys

p.Arg954Cys

p.Cys914Tyr

SPL

SPL

MS

SPL

MS-Cys

MS-Cys

MS-Cys

SPL

MS-Cys

NS

MS-Cys

MS-Cys

SPL

NS

NS

MS

MS-Cys

NS

NS

MS-Cys

MS-Cys

MS-Cys

n.p.

n.p.

+

+

+

+

+

+

n.p.

n.p.

n.p.

+

+

n.p.

+

+

+

+

n.p.

+

+

+

+

+

+

+

+

-

+

+

+

+

+

+

+

+

+

+

-

+

+

+

+

+

MFS

MFS

MFS

MFS

MFS

sMFS

MFS

MFS

MFS

MFS

MFS

MFS

MFS

MFS

MFS

MFS

sMFS

MFS

MFS

MFS

MFS

MFS

+

-

+

+

+

-

-

+

+

+

+

+

+

+

+

+

-

+

+

-

+

+

+

+

+

+

+

-

+

+

+

+

+

+

+

+

+

+

-

+

+

+

+

+

EL

EL

EL

EL

M

EL

EL

EL

EL

M

EL

EL

EL

M

EL

EL

M

M

M

EL

EL

EL

/

/

/

/

/

/

/

/

/

/

/

/

/

/

/

/

/

/

/

/

/

/

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

21

17

51

8

20

5

40

35

36

20

43

10

18

19

8

21

12

12

34

6

7

35

33

49

50

52

52

54

54

56

56

57

60

60

60

IVS61

62

63

63

64

65

#90

#91

#92

#93

#94

#95

#96

#97

#98

#99

#100

#101

#102

#103

#104

#105

#106

#107

c.8326C>T

c.8147dup

c.7999G>T

c.7897T>C

c.7774T>A

c.7699+1G>A

c.7532G>A

c.7498T>C

c.7487G>A

c.7071delC

c.6917G>A

c.6883T>C

c.6661T>C

c.6625G>T

c.6491G>A

c.6448C>T

c.6169C>T

c.6160C>T

c.5788+5G>A

p.Arg2776*

p.Tyr2716*

p.Glu2667*

p.Cys2633Arg

p.Cys2592Ser

p.?

p.Cys2511Tyr

p.Cys2500Arg

p.Cys2496Tyr

p.Val2358Serfs*40

p.Arg2306His

p.Cys2295Arg

p.Cys2221Arg

p.Glu2209*

p.Cys2164Tyr

p.Arg2150Cys

p.Arg2057*

p.Gln2054*

p.?

NS

FS

NS

MS-Cys

MS-Cys

SPL

MS-Cys

MS-Cys

MS-Cys

FS

MS

MS-Cys

MS-Cys

NS

MS-Cys

MS-Cys

NS

NS

SPL

+

+

+

n.p.

+

n.p.

+

+

n.p.

n.p.

n.p.

+

n.p.

+

+

+

+

n.p.

n.p.

+

+

-

-

+

+

+

+

+

-

-

-

+

+

+

+

+

+

+

MFS

MFS

sMFS

sMFS

MFS

MFS

MFS

MFS

MFS

sMFS

sMFS

sMFS

MFS

MFS

MFS

MFS

MFS

MFS

MFS

+

+

-

-

+

+

+

+

+

-

-

+

+

+

+

+

+

+

+

+

+

-

+

+

+

+

+

+

+

+

-

+

+

+

+

+

+

-

-

-

M

M

EL

EL

EL

/

M

M

M

EL

/

EL

-

EL

-

EL

EL

/

/

/

/

-

/

/

+

/

/

/

/

/

/

/

/

/

/

/

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

16

32

5

30

45

26

15

40

28

19

41

8

46

5

42

28

31

24

37

34

MASS phenotype. All variants have been previously reported as pathogenic, except for 2 VUS (MASS case #57, suspected MFS case #97, in italics). FBN1

A total of 54 previously described FBN1 different variants were identified in 58 patients with a clinical diagnosis of MFS or suspected MFS (sMFS) and 1

IVS46

#89

Ghent criteria (at visit)

-

-

-

-

-

-

-

+

#108

#109

#110

#111

#112

#113

#114

#115

MFS

ELS

sMFS

sMFS

sMFS

sMFS

sMFS

sMFS

Clinical Diagnosis (pre-test)

+

-

-

+

-

+

+

-

Systemic score

+

-

+

-

+

-

-

+

Cardiac

Patients with no detectable FBN1 mutation

Case

TABLE 3

-

EL

M

-

-

-

M

-

Ocular

NS: nonsense; FS: frameshift; SPL: splice site; GD: gross deletion

/

/

/

/

/

/

/

/

Nervous

-

-

-

-

-

-

-

-

Pulmonary

19

55

14

13

12

13

15

15

Age at genetic counseling (years)

-

-

rs25458

-

rs25458

rs363824

-

-

SNPs

-

-

c.1875T>C

-

c.1875T>C

c.6681A>C

-

-

Nucleotide change

-

-

p.Asn625=

-

p.Asn625=

p.Ser2227=

-

-

Protein change

35

the presence of spontaneous pneumothorax; (-) indicates the feature is absent. MS: missense; MS-Cys: missense introducing/eliminating a Cysteine residue;

involvement: (+) indicates the presence of dural ectasia; (-) indicates the feature is absent; (/) indicates unavailable data. Pulmonary involvement: (+) indicates

≥3.0 for subjects younger than 20 years; (-) indicates the feature is absent. Ocular involvement includes ectopia lentis (EL) and myopia (M). Nervous

) no systemic involvement (score <7). Cardiac involvement: (+) indicates aortic root enlargement (Z-score ≥2.0 for subjects aged 20 years and above, Z-score

are also reported. Ghent criteria: (+) indicates fulfillment and (-) indicates non-fulfillment. Systemic score: (+) indicates systemic involvement (score ≥7) and (-

of the same variant in the proband and other affected family members and/or absence in healthy family members; n.p.=not performed. Clinical signs of patients

reference sequences are: NM_000138.4, LRG_778t1, NG_008805.2. Coding exons are numbered from 1 to 65. Segregation studies: (+) indicates the presence

+

+

+

+

+

+

+

+

#117

#118

#119

#120

#121

#122

#123

#124

MFS

MFS

MFS

MFS

MFS

MFS

MFS

MFS

MFS

+

+

+

+

-

+

+

+

+

+

+

+

+

+

+

+

+

+

-

EL

-

M

EL

EL

-

M

-

/

/

/

/

/

/

/

/

/

-

-

-

-

-

-

-

-

-

22

5

30

42

21

34

20

26

56

-

rs25458

-

-

rs377338217

rs112287730

-

-

rs25458

-

c.1875T>C

-

-

c.4306G>A

c.2956G>A

-

-

c.1875T>C

-

p.Asn625=

-

-

p.Val1436Met

p.Ala986Thr

-

-

p.Asn625=

ectopia lentis; SNPs: single nucleotide polymorphisms; FBN1 reference sequence is NM_000138.4

36

absent. Unavailable data are indicated as (/). MFS: Marfan syndrome; sMFS: suspected Marfan syndrome; ELS: ectopia lentis syndrome; M: myopia; EL:

years; (-) indicates the feature is absent. Ocular involvement includes ectopia lentis (EL) and myopia (M). Pulmonary involvement: (-) indicates the feature is

<7). Cardiac involvement: (+) indicates aortic root enlargement (Z-score ≥2.0 for subjects aged 20 years and above, Z-score ≥3.0 for subjects younger than 20

indicates fulfillment and (-) indicates non-fulfillment. Systemic score: (+) indicates systemic involvement (score ≥7) and (-) no systemic involvement (score

Clinical signs of patients (n=17) negative to NGS and MLPA analyses are reported together with FBN1 polymorphisms identified. Ghent criteria: (+)

+

#116

Highlights

x x x x

Mutational screening of the FBN1 gene for the diagnosis of Marfan Syndrome (MFS) Detection rate for FBN1 variants in the study group is 86.3% (107/124 patients) FBN1 screening facilitates MFS diagnosis specially in young presymptomatic patients Multidisciplinary clinical evaluation improves MFS management and outcomes

37