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Expanding the genetic and phenotypic spectrum of ACTA2-related vasculopathies in a Dutch cohort

Open AccessPublished:September 01, 2022DOI:https://doi.org/10.1016/j.gim.2022.07.009

      Abstract

      Purpose

      Heterozygous pathogenic/likely pathogenic (P/LP) variants in the ACTA2 gene confer a high risk for thoracic aortic aneurysms and aortic dissections. This retrospective multicenter study elucidates the clinical outcome of ACTA2-related vasculopathies.

      Methods

      Index patients and relatives with a P/LP variant in ACTA2 were included. Data were collected through retrospective review of medical records using a standardized questionnaire.

      Results

      A total of 49 individuals from 28 families participated in our study. In total, 20 different ACTA2 variants were detected. Aortic events occurred in 65% of the cases (78.6% index patients and 47.6% relatives). Male sex and hypertension emerged as significantly associated with aortic events. Of 20 individuals, 5 had an aortic diameter of <45 mm (1.77 inches) at the time of the type A dissection. Mean age at first aortic event was 49.0 ± 12.4 years. Severe surgical complications for type A and type B dissection occurred in 25% and 16.7% of the cases and in-hospital mortality rates were 9.5% and 0%, respectively.

      Conclusion

      P/LP ACTA2 variants are associated with an increased risk for an aortic event and age-related penetrance, which emphasizes the importance of early recognition of the disease. Caregivers should be aware of the risk for aortic dissections, even in individuals with aortic diameters within the normal range.

      Keywords

      Introduction

      Pathogenic (P) variants in the ACTA2 gene (OMIM 102620, ORPHA:91387, ORPHA:2573), encoding the smooth muscle cell–specific isoform of alpha-actin, can cause a variety of vascular disorders including thoracic aortic aneurysms and dissections, premature coronary artery disease (CAD), and ischemic stroke.
      • Guo D.C.
      • Papke C.L.
      • Tran-Fadulu V.
      • et al.
      Mutations in smooth muscle alpha-actin (ACTA2) cause coronary artery disease, stroke, and Moyamoya disease, along with thoracic aortic disease.
      ,
      • Guo D.C.
      • Pannu H.
      • Tran-Fadulu V.
      • et al.
      Mutations in smooth muscle alpha-actin (ACTA2) lead to thoracic aortic aneurysms and dissections.
      In addition, P variants in ACTA2 are associated with congenital heart diseases (eg, bicuspid aortic valve [BAV] and patent ductus arteriosus), iris flocculi, and livedo reticularis. ACTA2-related vasculopathy is inherited as an autosomal dominant condition with reduced and age-related penetrance. Little is known about the risk factors for an aortic event and surgical outcome. Male sex, aortic diameter, and specific variants in ACTA2 are associated with higher risk for aortic events than the general population.
      • Guo D.C.
      • Pannu H.
      • Tran-Fadulu V.
      • et al.
      Mutations in smooth muscle alpha-actin (ACTA2) lead to thoracic aortic aneurysms and dissections.
      • Regalado E.S.
      • Guo D.C.
      • Prakash S.
      • et al.
      Aortic disease presentation and outcome associated with ACTA2 mutations.
      • Disabella E.
      • Grasso M.
      • Gambarin F.I.
      • et al.
      Risk of dissection in thoracic aneurysms associated with mutations of smooth muscle alpha-actin 2 (ACTA2).
      Although type A dissections occur more frequently in individuals with a P or likely P (LP) ACTA2 variant, type B dissections are present at younger age. Type B dissections are often complicated by aortic rupture and/or visceral or limb ischemia, for which surgical intervention is needed.
      • Regalado E.S.
      • Guo D.C.
      • Prakash S.
      • et al.
      Aortic disease presentation and outcome associated with ACTA2 mutations.
      The ACTA2 gene is located on chromosome 10 at q23.31 and consists of 8 coding exons that encode a 377 amino acid protein. Until October 2021, 39 different variants in the ACTA2 gene have been included in the Universal Mutation Database (www.umd.be). The vast majority (90%) are missense variants.
      In this retrospective multicenter study, we described the clinical presentation of 49 newly identified individuals from 28 families with a P/LP ACTA2 variant and elucidated the clinical outcome of ACTA2-related vasculopathies, including risk factors for an aortic event, detailed surgical outcomes, and survival.

      Materials and Methods

      Study population

      Index patients and relatives with a P/LP variant in the ACTA2 gene were included in this study at all 7 tertiary referral centers in the Netherlands from 2018 to 2020. Data were collected through retrospective review of available medical records using standardized questionnaire sent to involved clinical geneticists. This questionnaire addressed the genetic test results, the occurrence of thoracic aortic aneurysm and dissection (TAAD) and related treatments, various cardiovascular and ophthalmologic abnormalities, obstetric complications, other relevant medical conditions, and medication use.
      The Stanford classification was used to classify thoracic aortic dissections, type A dissection involves the ascending aorta/arch, whereas type B dissection originates in the descending thoracic aorta, distal to the left subclavian artery.
      • Daily P.O.
      • Trueblood H.W.
      • Stinson E.B.
      • Wuerflein R.D.
      • Shumway N.E.
      Management of acute aortic dissections.
      Absolute aortic diameters of ≥40 mm (1.57 inches) for the thoracic aorta and ≥30 mm (1.18 inches) for the abdominal aorta were classified as an aneurysm.
      • Hirsch A.T.
      • Haskal Z.J.
      • Hertzer N.R.
      • et al.
      ACC/AHA 2005 Practice Guidelines for the management of patients with peripheral arterial disease (lower extremity, renal, mesenteric, and abdominal aortic): a collaborative report from the American Association for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines (Writing Committee to Develop Guidelines for the Management of Patients With Peripheral Arterial Disease): endorsed by the American Association of Cardiovascular and Pulmonary Rehabilitation; National Heart, Lung, and Blood Institute; Society for Vascular Nursing; TransAtlantic Inter-Society Consensus and Vascular Disease Foundation.
      ,
      • Verhagen J.M.A.
      • Kempers M.
      • Cozijnsen L.
      • et al.
      Expert consensus recommendations on the cardiogenetic care for patients with thoracic aortic disease and their first-degree relatives.
      An aortic event was defined as an elective aortic aneurysm repair or an aortic dissection, whether or not preceded by an aneurysm. The z-score was calculated using the web-based calculation tool on the Marfan foundation site (https://marfan.org/dx/z-score-adults/), which uses the normograms of Devereux et al
      • Devereux R.B.
      • de Simone G.
      • Arnett D.K.
      • et al.
      Normal limits in relation to age, body size and gender of two-dimensional echocardiographic aortic root dimensions in persons ≥15 years of age.
      and the method of Dubois and Dubois
      • Du Bois D.
      • Du Bois E.F.
      A formula to estimate the approximate surface area if height and weight be known.
      to calculate the body surface area.
      Systolic blood pressure of >130 mm Hg and/or diastolic blood pressure of >80 mm Hg and/or the use of antihypertensive agents was defined as hypertension.
      • Whelton P.K.
      • Carey R.M.
      • Aronow W.S.
      • et al.
      2017 ACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA guideline for the prevention, detection, evaluation, and management of high blood pressure in adults: A report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice guidelines.
      Severe complications implicated those that are potentially life threatening or are disabling for the individual.

      Variant analysis

      All index patients underwent sequencing of a TAAD gene panel, including at least the definitive and strong genes for TAAD.
      • Renard M.
      • Francis C.
      • Ghosh R.
      • et al.
      Clinical validity of genes for heritable thoracic aortic aneurysm and dissection.
      The pathogenicity of variants in the ACTA2 gene (NG_011541.1, NM_1613.3) was assessed using (Alamut Visual v2.11) software, which integrates data from large-scale population genetic studies, evolutionary conservation of nucleotides and amino acids, and in silico missense and messenger RNA (mRNA) splicing prediction tools. The Mendelian Clinically Applicable Pathogenicity Score was added to improve classification of rare missense variants.
      • Jagadeesh K.A.
      • Wenger A.M.
      • Berger M.J.
      • et al.
      M-CAP eliminates a majority of variants of uncertain significance in clinical exomes at high sensitivity.
      A threshold of 0.025 correctly dismisses 60% of variants of uncertain significance at 95% sensitivity.
      ACTA2 variants with a minor allele frequency of >1% in the Genome Aggregation Database v2.1.1
      • Walsh R.
      • Thomson K.L.
      • Ware J.S.
      • et al.
      Reassessment of Mendelian gene pathogenicity using 7,855 cardiomyopathy cases and 60,706 reference samples.
      and synonymous and intronic variants without predicted effect on mRNA splicing were excluded from further analysis. If possible, cosegregation of the variant with the disease phenotype in the family was analyzed. RNA sequencing on RNA isolated from fibroblasts obtained from skin biopsies was performed for the splice variant p.[Ala206Gly,Glu207_Ile269del] in the last exon of ACTA2. Variants were interpreted according to the 2015 American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines
      • Richards S.
      • Aziz N.
      • Bale S.
      • et al.
      Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
      and classified into 5 categories ie, P, LP, uncertain significance, likely benign, and benign. Only variants classified as P or LP were included in this study.
      Of note, de novo ACTA2 variants disrupting the Arg179 residue are associated with a specific phenotype referred to as multisystemic smooth muscle dysfunction syndrome (OMIM 613834, ORPHA:404463) and were excluded from this study.

      Statistical analysis

      Continuous variables were summarized by mean and SD. Categorical variables are shown in frequencies and percentages. Comparisons were made using the Student t test and χ2 test with Yates’ correction, respectively. A P value of <.05 was considered statistically significant. A binary logistic regression analysis was used to identify risk factors associated with aortic event. Variables with a P value around or <.05 in the univariate analysis were included in the multiple logistic regression model. Odds ratios, CIs, and P values were used for interpretation.

      Results

      Characteristics of the study population

      A total of 49 individuals from 28 families were included. The number of men and women were almost equal (Table 1). Median age at genetic diagnosis was 50.0 (interquartile range = 25.0) years.
      Table 1Clinical characteristics
      VariableAll (N = 49)With Aortic Event (n = 32)Without Aortic Event (n = 16)Unknown Aortic Event (n = 1)P value
      Median age at diagnose, y (IQR)50.0 (25.0)50.5 (20.3)45.00 (33.0).754
      Sex1.051
       Male23194
       Female261312
      Blood pressure.005
       Hypertension22175
       No hypertension19811
       Unknown871
      BMI, mean (SD)27.7 (4.0)28.2 (7.3)27.2 (3.7).354
      Smoking history1.507
       Smoking22148
       No smoking1495
       Unknown1393
      BMI, body mass index; IQR, interquartile range.

      Molecular studies

      A total of 20 different P/LP ACTA2 variants were detected in this cohort, including 18 missense, 1 splice site, and 1 frameshift variant (Figure 1, Table 2). Functional assay for the splice variant p.[Ala206Gly,Glu207_Ile269del] showed no aberrant splicing and an equal presence of both alleles. The frameshift variant p.(Ser340Cysfs∗26) is a deletion of 2 nucleotides, leading to a frameshift and a premature protein termination in the last exon. In total, 8 variants were novel. A total of 4 variants were found in more than 1 family; p.(Arg39Cys) was found in 3 families and p.(Arg149Cys), p.(His175Asp), and p.(Arg208His) were each found in 2 families. In 17 families, 63 relatives were screened for the familial P/LP ACTA2 variants, including 30 heterozygotes and 33 nonheterozygotes of the P/LP variants in ACTA2. Medical records of 21 heterozygotes from 11 families were available: 16 first-degree and 5 second-degree relatives. Relatives from 6 of the 17 families were not included because they live abroad, their test result was unknown at the time of inclusion, or none of the relatives were heterozygotes of the P/LP variants in ACTA2.
      Figure thumbnail gr1
      Figure 1Schematic representation of the ACTA2 gene. Schematic overview of the ACTA2 gene, with the location of pathogenic and likely pathogenic variants in ACTA2 found in our cohort. The variants displayed above the exons are novel and the variants displayed below the exons are known. Orange boxes represent coding exons, gray boxes represent untranslated region (UTR), and connecting lines represent introns (unscaled).
      Table 2Overview of pathogenic and likely pathogenic ACTA2 variants identified in this study
      Nucleotide ChangeProtein ChangeCoding EffectMAF gnomADM-CAP ScoreEvidence
      • Richards S.
      • Aziz N.
      • Bale S.
      • et al.
      Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
      ,
      PS4 is given after calculating BayesianOR according to the recommendation of Kyung Cho et al (Cho YK, Won D, Keum C, Lee BH, Seo GH, Lee BC. A novel PS4 criterion approach based on symptoms of rare diseases and in-house frequency data in a Bayesian framework. https://doi.org/10.1101/2020.07.22.215426). Odds ratios are > 5.0 and CI does not include 1.0.
      ,
      PM2 is used as a supportive argument according to the latest recommendations, ClinGen Sequence Variant Interpretation Recommendation for PM2 version 1.0
      ,
      Specification of PP2: the missense constraint score (z-score ≥ 3.09) from gnomAD was used.19,20 z-score for ACTA2 was 3.2 (https://gnomad.broadinstitute.org); the missense variants were spread over all exons.
      ,
      Specification for PP4: presence of iris flocculi, which is highly specific for ACTA2 related vasculopathies.
      ClassificationKnown/Novel
      c.13G>Cp.(Glu5Gln)Missense1/313900.416PS4, PM2(s), PP2, PP1Likely pathogenicDescribed previously
      • Overwater E.
      • Marsili L.
      • Baars M.J.H.
      • et al.
      Results of next-generation sequencing gene panel diagnostics including copy-number variation analysis in 810 patients suspected of heritable thoracic aortic disorders.
      c.46T>Cp.(Ser16Pro)MissenseAbsent0.765PS4, PM2(s), PP1, PP2, PP3Likely pathogenicNovel
      c.101C>Gp.(Pro34Arg)MissenseAbsent0.692PS4, PM2(s), PP1, PP2, PP3Likely pathogenicNovel
      c.115C>Tp.(Arg39Cys)Missense1/313740.347PS4, PM2(s), PM5, PP2, PP3, PP5PathogenicKnown
      • Hoffjan S.
      • Waldmüller S.
      • Blankenfeldt W.
      • et al.
      Three novel mutations in the ACTA2 gene in German patients with thoracic aortic aneurysms and dissections.
      c.296C>Gp.(Ala99Gly)Missense5/2510080.389PS4, PM2(s), PP1, PP2, PP3Likely pathogenicNovel
      c.419C>Tp.(Ala140Val)MissenseAbsent0.592PS1, PS4, PM2(s), PP2, PP3, PP5PathogenicKnown
      • Lerner-Ellis J.P.
      • Aldubayan S.H.
      • Hernandez A.L.
      • et al.
      The spectrum of FBN1, TGFβR1, TGFβR2 and ACTA2 variants in 594 individuals with suspected Marfan syndrome, Loeys-Dietz syndrome or thoracic aortic aneurysms and dissections (TAAD).
      c.445C>Tp.(Arg149Cys)Missense1/2787320.509PS1, PS4, PM2(s), PM5, PM6, PP2, PP3, PP4, PP5PathogenicKnown
      • Guo D.C.
      • Pannu H.
      • Tran-Fadulu V.
      • et al.
      Mutations in smooth muscle alpha-actin (ACTA2) lead to thoracic aortic aneurysms and dissections.
      c.485C>Ap.(Thr162Asn)MissenseAbsent0.550PS4, PM2(s), PP1, PP2, PP3, PP4Likely pathogenicNovel
      c.523C>Gp.(His175Asp)MissenseAbsent0.525PS4, PM2(s), PP1, PP2, PP3Likely pathogenicNovel
      c.526G>Ap.(AIa176Thr)Missense1/2510620.288PS4, PM2(s), PP1, PP2, PP3Likely pathogenicNovel
      c.587C>Ap.(Thr196Asn)MissenseAbsent0.194PS4, PM2(s), PP1, PP2, PP3Likely pathogenicNovel
      c.607G>Cp.(Val203Leu)Missense1/2509300.082PS4, PM2(s), PP1, PP2Likely pathogenicDescribed previously
      • Overwater E.
      • Marsili L.
      • Baars M.J.H.
      • et al.
      Results of next-generation sequencing gene panel diagnostics including copy-number variation analysis in 810 patients suspected of heritable thoracic aortic disorders.
      c.623G>Ap.(Arg208His)Missense3/2505720.559PS4, PM2(s), PP1, PP2, PP3Likely pathogenicNovel
      c.772C>Tp.(Arg258Cys)Missense1/313840.516PS1, PS4, PM2, PM5, PP2, PP3, PP5PathogenicKnown
      • Guo D.C.
      • Papke C.L.
      • Tran-Fadulu V.
      • et al.
      Mutations in smooth muscle alpha-actin (ACTA2) cause coronary artery disease, stroke, and Moyamoya disease, along with thoracic aortic disease.
      c.808G>Ap.[Ala206Gly,Glu207_Ile269del]Splice1/2508200.696PS3, PS4, PM2(s), PP1, PP4, PP5PathogenicKnown
      • Regalado E.S.
      • Guo D.C.
      • Prakash S.
      • et al.
      Aortic disease presentation and outcome associated with ACTA2 mutations.
      c.835A>Gp.(Thr279Ala)MissenseAbsent0.100PS1, PS4, PM2(s), PP2PathogenicDescribed previously
      • Overwater E.
      • Marsili L.
      • Baars M.J.H.
      • et al.
      Results of next-generation sequencing gene panel diagnostics including copy-number variation analysis in 810 patients suspected of heritable thoracic aortic disorders.
      c.854T>Cp.(Met285Thr)Missense1/2511060.367PS1, PS4, PM2(s), PP2, PP3PathogenicDescribed previously
      • Overwater E.
      • Marsili L.
      • Baars M.J.H.
      • et al.
      Results of next-generation sequencing gene panel diagnostics including copy-number variation analysis in 810 patients suspected of heritable thoracic aortic disorders.
      c.977C>Ap.(Thr326Asn)Missense15/2508860.141PS4, PM2(s), PP1, PP2, PP5Likely pathogenicKnown
      • Guo D.C.
      • Papke C.L.
      • Tran-Fadulu V.
      • et al.
      Mutations in smooth muscle alpha-actin (ACTA2) cause coronary artery disease, stroke, and Moyamoya disease, along with thoracic aortic disease.
      c.1019_1020delCTp.(Ser340Cysfs∗26)FrameshiftAbsentN/APS1, PS4, PM2(s), PP5PathogenicKnown
      • Renard M.
      • Callewaert B.
      • Baetens M.
      • et al.
      Novel MYH11 and ACTA2 mutations reveal a role for enhanced TGFβ signaling in FTAAD.
      c.1120C>Tp.(Arg374Cys)Missense1/2512420.275PS1, PS4, PM2(s), PP2, PP3PathogenicDescribed previously
      • Overwater E.
      • Marsili L.
      • Baars M.J.H.
      • et al.
      Results of next-generation sequencing gene panel diagnostics including copy-number variation analysis in 810 patients suspected of heritable thoracic aortic disorders.
      NCBI reference sequence: NG_011541.1, NM_001613.3.
      ClinGen, Clinical Genome Resource; gnomAD, Genome Aggregation Database; MAF, minor allele frequency; M-CAP, Mendelian Clinically Applicable Pathogenicity; N/A, not applicable.
      a PS4 is given after calculating BayesianOR according to the recommendation of Kyung Cho et al (Cho YK, Won D, Keum C, Lee BH, Seo GH, Lee BC. A novel PS4 criterion approach based on symptoms of rare diseases and in-house frequency data in a Bayesian framework. https://doi.org/10.1101/2020.07.22.215426). Odds ratios are > 5.0 and CI does not include 1.0.
      b PM2 is used as a supportive argument according to the latest recommendations, ClinGen Sequence Variant Interpretation Recommendation for PM2 version 1.0
      c Specification of PP2: the missense constraint score (z-score ≥ 3.09) from gnomAD was used.
      • Lek M.
      • Karczewski K.J.
      • Minikel E.V.
      • et al.
      Analysis of protein-coding genetic variation in 60,706 humans.
      ,
      • Ellard S.
      • Baple E.L.
      • Callaway A.
      • et al.
      ACGS Best Practice Guidelines for Variant Classification in Rare Disease 2020. Association for Clinical Genomic Science.
      z-score for ACTA2 was 3.2 (https://gnomad.broadinstitute.org); the missense variants were spread over all exons.
      d Specification for PP4: presence of iris flocculi, which is highly specific for ACTA2 related vasculopathies.
      No proven de novo variants were found, but not all parents were available for genetic testing.

      Aortic events

      In total, 32 of 49 (65.0%) individuals had an aortic event, including elective aortic aneurysm repair in 4 and aortic dissection in 29 (20 type A, 12 type B, and 2 abdominal) (Table 3). Three individuals underwent elective aortic aneurysm repair because of ascending aortic aneurysms of diameters of 50, 50, and 60 mm (1.97, 1.97, and 2.36 inches, respectively) and 1 for an abdominal aortic aneurysm of 51 mm (2.01 inches). A total of 5 individuals had both type A and type B dissection, with several years between the events and 1 had an elective aortic aneurysm repair followed by a type A dissection years later. Mean age at first aortic event was 49.0 (±12.4) years. The youngest individual was a 24-year old male with a type B dissection extending from the aortic arch to the renal arteries. Of the 18 individuals without an aortic event, 9 had an aortic aneurysm, including 3 at the level of the aortic root, 5 on the ascending aorta, and 1 was abdominal.
      Table 3Surgical data
      VariableElective Aortic Aneurysm Repair (n = 4)Type A Dissection (n = 20)Type B Dissection (n = 12)Dissection Abdominal Aorta (n = 2)
      Operation performed (% of total)4 (100)17 (85.0)6 (50.0)1 (50.0)
      Mean diameter, mm (SD)47.2 (15.1)
      1.86 inches
      33 (7.1)
      1.30 inches
      70
      2.76 inches
      Type of intervention (% of operations)
       SCAR1 (25)
       SCAR and hemiarch replacement1 (25)9 (52.9)
       SCAR and aortic arch replacement1 (25)1 (5.9)
       AVR plus ascending aorta and hemiarch replacement5 (29.4)
       AVR plus ascending aorta and arch replacement2 (11.8)
       Descending aortic replacement1 (16.7)
       TEVAR procedure5 (83.3)
       EVAR procedure1 (25)
       Aortic bifurcation prosthesis1 (100%)
      Number of patients with severe complications (%)1 (25)4 (25)1 (16.7)
      Severe complications
       Death2
       Sternum infection1
       Perioperative rupture of descending aorta1
       Myocardial infarction1
       Cardiac tamponade1
      AVR, aortic valve replacement; EVAR, endovascular aortic repair; TEVAR, thoracic endovascular aortic repair; SCAR, supracoronary ascending aorta replacement.
      a 1.86 inches
      b 1.30 inches
      c 2.76 inches
      The mean age at the time of diagnosis of the aortic aneurysm was 50.9 (± 10.4) years. The mean diameter of the thoracic aortic aneurysms was 46.0 (± 4.3) mm (1.81 [± 0.17] inches) and the diameter of the abdominal aortic aneurysm was 35 mm (1.38 inches). In total, 8 individuals were asymptomatic, which is defined as the absence of an aortic event and/or an aortic aneurysm. The mean diameter in asymptomatic individuals was 30.6 (20-38) mm (1.20 [0.79-1.5] inches) and the mean age was 37.0 (± 17.0) years. One individual was a post mortem diagnosed heterozygote; no information about aortic diameters was present.
      Excluding the index patients, 16 of 21 (76.2%) relatives with a P/LP variant in ACTA2 had a phenotype, including 10 (47.6%) with an aortic event at a mean age of 53.3 (±12.8) years, 3 with an aortic aneurysm, 2 with iris flocculi, and 1 with premature CAD. The 5 relatives without signs of the disease had a mean age of 53.4 (±18.4) years.
      The mean diameter of the ascending aorta at the time of an aortic dissection was 47.2 (30-79) mm (1.86 [1.18- 3.11] inches) for a type A dissection based on available data from 9 individuals. Five of them, in 4 females and 1 male, had a diameter of ≤45 mm (1.77 inches) at the time of dissection, namely 30 mm (1.18 inches) (z-score = –1.3), 30 mm (1.18 inches) (z-score = –0.9), 40 mm (1.57 inches) (z-score = 3.0), 41 mm (1.61 inches) (z-score = 2.9), and 45 mm (1.77 inches) (z-score = 3.5). The diameter of the aorta at the time of a type B dissection was known for 2 patients: 28 (1.10 inches) (z-score = –2.47) mm and 38 mm (1.50 inches) (z-score = 0.4). The diameter of the abdominal aorta at the time of dissection in 1 patient was 70 mm (2.76 inches) (z-score = 12.5). In the 4 individuals with an elective aortic aneurysm repair, the diameter at the time of surgery was 50 mm (1.97 inches), 50 mm (1.97 inches), and 60 mm (2.36 inches) for the ascending aorta and 51 mm (2.01 inches) for the abdominal aorta. In most individuals (17/28) with a thoracic aortic aneurysm, the maximum diameter was found at the level of the ascending aorta.
      Assessment of the cardiovascular risk factors revealed that the prevalence of aortic events was higher in men than in women (P = .051) and significantly higher in individuals with hypertension than in individuals without hypertension before dissection (P = .005) (Table 1). Hypertension (odds ratio = 9.768, CI = 1.701-56.076, P = .011) and male sex (odds ratio = 8.526, CI = 1.412-51.493, P = .020) were identified as independent risk factors for the occurrence of an aortic event in the multivariable analysis. No correlation was found between sex and hypertension (P = .366). For 8 individuals, blood pressure measurements were not available. For 2 individuals, no medical records were available; they were diagnosed as heterozygotes post mortem. Other cardiovascular risk factors like body mass index and smoking did not significantly increase the risk for an aortic event (Table 1).

      Survival

      In total, 8 individuals died at a mean age of 51.5 years (±10.6 years), including 6 deaths caused by an aortic event and in 2 the cause of death was unknown. Three individuals died before surgical intervention for a type A dissection could be performed, including 1 post mortem diagnosed heterozygote. One individual underwent a supracoronary ascending aorta replacement (SCAR) plus hemiarch and died 1 month later because of a new acute type B dissection. Two individuals died as a result of complications of the surgery. One individual first had an SCAR with replacement of the aortic arch because of a type A dissection extending till below the renal arteries and died in hospital after a second surgery for the remaining dissection. The other received aortic valve replacement with aortic ascendens and arch replacement because of an acute type A dissection. After the operation, a myocardial infarction occurred caused by an occlusion of the left anterior descending artery. Because of hemodynamic instability, extracorporeal membrane oxygenation was needed. The individual died of cardiogenic shock after explant of the extracorporeal membrane oxygenation.

      Surgical data

      In total, 17 individuals (85.0%) with a type A dissection underwent surgery (Table 3). Severe complications occurred in 4 (25%) of the type A dissection surgeries, including 2 deaths as a result of multiple complications as described before. One individual suffered from a sternum infection and 1 had a myocardial infarction.
      In total, 6 individuals (50.0%) with a type B dissection underwent surgery, including 5 with a thoracic endovascular aortic repair (TEVAR) procedure. One individual had 7 years of medical treatment for a type B dissection, and after an elective ascending aortic and aortic arch replacement due to an aneurysm, the descending aorta was replaced by a prosthesis a few months later (Table 3). A total of 4 individuals had a TEVAR immediately after the type B dissection was diagnosed, in 3 because of an acute ischemic leg and in 1 because of a small rupture in the aortic arch. In 1 individual, a TEVAR was performed 9 days after the type B dissection was diagnosed; information about the reason for surgical intervention could not be obtained.
      Severe complications occurred in one (16.7%) of the surgeries for type B dissection (Table 3). This individual had a perioperative rupture of the descending aorta during the TEVAR procedure with paraplegia afterward. Hemodynamic instability occurred immediately after the procedure due to a rupture, requiring a lateral thoracotomy.
      Two individuals had surgeries for a consecutive type A and B dissection and type B and A dissection, respectively. One individual received aortic bifurcation prosthesis because of an abdominal aortic dissection. In total, 5 individuals were treated conservatively for a type B dissection and 1 for an abdominal aortic dissection.
      A total of 4 individuals underwent elective aortic aneurysm repair. The SCAR plus hemiarch replacement was complicated by cardiac tamponade. The individual who underwent the endovascular aortic repair procedure underwent a secondary intervention after 9 years because of an endoleak. The individual with an SCAR, underwent an aortic valve replacement plus ascending aorta and arch replacement procedure because of a type A aortic dissection a few years later.

      Congenital heart malformations

      In total, 45 individuals were examined by a cardiologist: 6 (13%) had a congenital heart malformation, including persistent ductus arteriosus (PDA) in 3, BAV in 2, and atrial septum defect type II in 1. These 6 individuals all had different P/LP variants in ACTA2 (Table 4). Four individuals were not examined by a cardiologist; 2 of them were obligate heterozygotes and 2 individuals were diagnosed as heterozygotes post mortem.
      Table 4Other health conditions in heterozygotes of pathogenic or likely pathogenic variant in ACTA2
      VariableACTA2 VariantFrequency (% of Total Patients Screened)
      Congenital heart malformation6 (13.3)
       PDAp.(Arg39Cys)3 (6.7)
      p.(His175Asp)
      p.(Ser16Pro)
       BAVp.(Glu5Gln)2 (4.4)
      p.(Met285Thr)
       ASD type IIp.(Ser340Cysfs∗26)1 (2.2)
      Eye anomalies10 (40)
       Iris flocculip.(Arg149Cys)7 (28)
      p.[Ala206Gly,Glu207_Ile269del]
      p.(Thr162Asn)
       Tortuosity retinal vesselsp.(Arg39Cys)1 (4)
       Keratoconusp.[Ala206Gly,Glu207_Ile269del]1 (4)
       Retinal tearp.(AIa176Thr)1 (4)
      ASD, atrium septum defect; BAV, bicuspid aortic valve; PDA, persistent ductus arteriosus.

      Ocular abnormalities

      Ophthalmologic examination was performed in 25 individuals and showed abnormalities in 10 (40%), of which 7 individuals had iris flocculi. All heterozygotes of the p.(Arg149Cys) variant in ACTA2 with ophthalmologic investigation (n = 5) had iris flocculi (Table 4). Other ocular abnormalities, including tortuosity of the retinal vessels, keratoconus, and retinal tear, were described once.

      Other clinical manifestations

      Occlusive vascular diseases were reported in 9 individuals, including CAD in 6, livedo reticularis in 2, and an ischemic cerebrovascular accident (CVA) in 1. Two individuals suffered from venous thrombosis.
      In total, 11 individuals underwent imaging of the coronary arteries, including computed tomography angiography in 8, coronary angiogram in 2, and myocardial perfusion imaging in 1. A myocardial infarction was diagnosed in 3, dissection of the left anterior descending artery in 1, CAD in 1, and stenosis of the right coronary artery in 1. All 3 myocardial infarctions were caused by atheromatosis. Two individuals underwent coronary artery bypass graft and 2 underwent percutaneous coronary intervention. Of the 6 individuals with CAD, 2 were younger than 45 years.
      Imaging of the cerebral arteries by computed tomography angiography or magnetic resonance angiography was performed in 16 individuals without neurologic symptoms and revealed abnormalities in 1 (6%), namely a dilated and tortuous course of the distal vertebral artery.
      Other vascular abnormalities included dilatation and elongation of the iliac arteries and tortuosity of the renal artery, each reported once. However, only 14 individuals underwent imaging of cerebral, thoracic, and abdominal vascular tree.
      An umbilical hernia was reported in 4 unrelated individuals, including 1 with an umbilical and inguinal hernia. Two related individuals had an inguinal hernia only. In 1 individual, the correction of an umbilical hernia with a mesh implantation was complicated by a perforation of the sigmoid, requiring reoperation.
      The obstetric history was available for 22 women with a total of 52 pregnancies. One individual underwent a cesarean section at 38 weeks of pregnancy because of resistant hypertension. Five days postpartum, a type A dissection occurred. The diameter of the ascending aorta was <40 mm (1.57 inches) and the sinotubular junction was 25 mm (0.98 inches) at the time of the valve sparing aortic replacement.

      Discussion

      We provide detailed phenotypic and genotypic data of 49 individuals from 28 families harboring 20 different heterozygous P/LP ACTA2 variants. We have identified 8 novel ACTA2 variants. There is no mutational hotspot because the P/LP variants found in our cohort were distributed all over the gene, in concordance with previous studies. Most P/LP variants in ACTA2 found in our cohort were missense variants. Only 1 frameshift in the last exon of ACTA2 and 1 splice site variant were identified.
      Guo et al
      • Guo D.C.
      • Pannu H.
      • Tran-Fadulu V.
      • et al.
      Mutations in smooth muscle alpha-actin (ACTA2) lead to thoracic aortic aneurysms and dissections.
      suggested a dominant negative disease mechanism in ACTA2-related vasculopathies. In smooth muscle cells of individuals with a P/LP ACTA2 variant, reduced ACTA2-containing fibers were shown, suggesting disruption of actin fiber assembly or stability. Renard et al
      • Renard M.
      • Callewaert B.
      • Baetens M.
      • et al.
      Novel MYH11 and ACTA2 mutations reveal a role for enhanced TGFβ signaling in FTAAD.
      described 2 nonsense variants in ACTA2, which suggested that premature truncating variants in ACTA2 can cause TAAD as well. Although no functional assays were performed, both variants are predicted to escape from nonsense-mediated mRNA decay because they are located at the end of ACTA2 and may still exert a dominant negative effect. The functional assay for the splice variant performed in our study supported the suggestion of a dominant negative effect. No functional assay has been performed for the frameshift variant, but this variant is also located in the last exon of ACTA2, therefore, a dominant negative effect seems plausible. The other novel variants identified in this study are all missense variants.
      An aortic event occurred in 65.0% the total study cohort. After the exclusion of index patients, 47.6% of family members had an aortic event at a mean age of 53.3 years (±12.8 years), which probably better represents the penetrance for aortic events of ACTA2 variants. Nevertheless, our family-based approach, starting from symptomatic individuals, will potentially result in an overestimation of the penetrance of disease-causing variants. An overall penetrance of 76% for aortic events at the age of 85 years has been described previously in a study with a similar approach in identifying heterozygotes of a P/LP ACTA2 variant.
      • Regalado E.S.
      • Guo D.C.
      • Prakash S.
      • et al.
      Aortic disease presentation and outcome associated with ACTA2 mutations.
      When including all phenotypical features of ACTA2 related vasculopathy, the penetrance of ACTA2 variants in relatives was 76.2%. The penetrance of ACTA2 variants in asymptomatic individuals without a family history of aortic disease, such as secondary or incidental findings from genetic testing, might be significantly lower.
      • Turner H.
      • Jackson L.
      Evidence for penetrance in patients without a family history of disease: a systematic review.
      Assessment of known cardiovascular risk factors revealed that the presence of hypertension significantly increased the risk for an aortic event (P = .011). Hypertension has not been described before as a specific risk factor for aortic events in heterozygotes of a P/LP variant in ACTA2. In individuals without a genetic predisposition, hypertension is a well-known risk factor for thoracic aortic aneurysms and aortic dissections.
      • Howard D.P.J.
      • Sideso E.
      • Handa A.
      • Rothwell P.M.
      Incidence, risk factors, outcome and projected future burden of acute aortic dissection.
      ,
      • Landenhed M.
      • Engström G.
      • Gottsäter A.
      • et al.
      Risk profiles for aortic dissection and ruptured or surgically treated aneurysms: a prospective cohort study.
      Therefore, it is important to treat all hypertensive ACTA2 individuals with medication that reduces hemodynamic stress and provide lifestyle advices. In a recent consensus statement for the management of individuals with P ACTA2 variants, it was even proposed that antihypertensive medication should be considered in all ACTA2 individuals, ie, also in normotensive individuals.
      • van de Laar I.M.B.H.
      • Arbustini E.
      • Loeys B.
      • et al.
      European reference network for rare vascular diseases (VASCERN) consensus statement for the screening and management of patients with pathogenic ACTA2 variants.
      Male sex also seems to be a predisposing factor for the occurrence of an aortic event in heterozygotes of a P/LP variant in ACTA2, as was reported previously by Regalado et al.
      • Regalado E.S.
      • Guo D.C.
      • Prakash S.
      • et al.
      Aortic disease presentation and outcome associated with ACTA2 mutations.
      This was also seen in individuals with Marfan syndrome caused by P variants in the FBN1 gene.
      • Détaint D.
      • Faivre L.
      • Collod-Beroud G.
      • et al.
      Cardiovascular manifestations in men and women carrying a FBN1 mutation.
      ,
      • Roman M.J.
      • Rosen S.E.
      • Kramer-Fox R.
      • Devereux R.B.
      Prognostic significance of the pattern of aortic root dilation in the Marfan syndrome.
      The underlying mechanism for this gender difference is still unclear and warrants further study.
      The vast majority of the individuals with a type A dissection underwent surgical intervention and half of the type B dissections were treated surgically. The type of surgeries performed in our cohort did not differ from the standard procedure for aortic dissections. Mortality rate in individuals without a known genetic predisposition for TAAD and with Marfan syndrome treated surgically for an acute type A dissection was 16.7% to 18% and treated with endovascular management for an acute type B dissection was 7.4% to 12.3%.
      • Trimarchi S.
      • Nienaber C.A.
      • Rampoldi V.
      • et al.
      Contemporary results of surgery in acute type A aortic dissection: the International Registry of Acute Aortic Dissection experience.
      • Pape L.A.
      • Awais M.
      • Woznicki E.M.
      • et al.
      Presentation, diagnosis, and outcomes of acute aortic dissection: 17-year trends from the International Registry of Acute Aortic Dissection.
      • Harky A.
      • Chan J.S.K.
      • Wong C.H.M.
      • Francis N.
      • Grafton-Clarke C.
      • Bashir M.
      Systematic review and meta-analysis of acute type B thoracic aortic dissection, open, or endovascular repair.
      • Evangelista A.
      • Isselbacher E.M.
      • Bossone E.
      • et al.
      Insights from the International Registry of Acute Aortic Dissection: A 20-year experience of collaborative clinical research.
      For heterozygotes of a P/LP variant in ACTA2 with an acute dissection, Regalado et al
      • Regalado E.S.
      • Guo D.C.
      • Prakash S.
      • et al.
      Aortic disease presentation and outcome associated with ACTA2 mutations.
      found an in-hospital mortality rate of 14%, but no details about the complications after surgical intervention were available. In our cohort, the in-hospital mortality rate after surgery for a type A dissection was 12.5% and for patients with endovascular treatment for a type B dissection was 0%. Therefore, it seems that individuals with a P/LP variant in ACTA2 do not have a higher in-hospital mortality rate after aortic dissection intervention than the individuals without a genetic predisposition for TAAD. The general consensus is to avoid a TEVAR procedure in individuals with genetic aortopathies, except in case of a complicated type B dissection. Our data can support the general consensus to perform a TEVAR procedure for complicated type B dissections in individuals with a P/LP variant in ACTA2 as in individuals with other genetic aortopathies.
      • van de Laar I.M.B.H.
      • Arbustini E.
      • Loeys B.
      • et al.
      European reference network for rare vascular diseases (VASCERN) consensus statement for the screening and management of patients with pathogenic ACTA2 variants.
      ,
      • Erbel R.
      • Aboyans V.
      • Boileau C.
      • et al.
      2014 ESC Guidelines on the diagnosis and treatment of aortic diseases: document covering acute and chronic aortic diseases of the thoracic and abdominal aorta of the adult. The Task Force for the Diagnosis and Treatment of Aortic Diseases of the European Society of Cardiology (ESC).
      According to current literature for individuals with P/LP ACTA2 variants, elective aortic root replacement should be considered in asymptomatic patients with a maximal aortic diameter between 45 and 50 mm (1.77 and 1.97 inches, respectively).
      • Howard D.P.J.
      • Sideso E.
      • Handa A.
      • Rothwell P.M.
      Incidence, risk factors, outcome and projected future burden of acute aortic dissection.
      ,
      • Carino D.
      • Agostinelli A.
      • Molardi A.
      • Benassi F.
      • Gherli T.
      • Nicolini F.
      The role of genetic testing in the prevention of acute aortic dissection.
      The American Heart Association (AHA) recommends elective surgery at a maximal ascending aortic diameter of 45 mm (1.77 inches).
      • Hiratzka L.F.
      • Bakris G.L.
      • Beckman J.A.
      • et al.
      2010 ACCF/AHA/AATS/ACR/ASA/SCA/SCAI/SIR/STS/SVM guidelines for the diagnosis and management of patients with thoracic aortic disease: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines, American Association for Thoracic Surgery, American College of Radiology, American Stroke Association, Society of Cardiovascular Anesthesiologists, Society for Cardiovascular Angiography and Interventions, Society of Interventional Radiology, Society of Thoracic Surgeons, and Society for Vascular Medicine.
      Although the diameter of the ascending aorta is a well-established risk factor for dissection, an interesting finding in our study is that more than half (55%) of the individuals had a diameter of ≤45 mm (1.77 inches) at the time of dissection. In literature, among 47 individuals with a P/LP variant in ACTA2 whose aortic diameters were available, 9 (19%) individuals had a diameter of ≤45 mm (1.77 inches) at the time of aortic dissection.
      • Guo D.C.
      • Pannu H.
      • Tran-Fadulu V.
      • et al.
      Mutations in smooth muscle alpha-actin (ACTA2) lead to thoracic aortic aneurysms and dissections.
      • Regalado E.S.
      • Guo D.C.
      • Prakash S.
      • et al.
      Aortic disease presentation and outcome associated with ACTA2 mutations.
      • Disabella E.
      • Grasso M.
      • Gambarin F.I.
      • et al.
      Risk of dissection in thoracic aneurysms associated with mutations of smooth muscle alpha-actin 2 (ACTA2).
      ,
      • Delsart P.
      • Vanlerberghe C.
      • Juthier F.
      • et al.
      The natural history of a family with aortic dissection associated with a novel ACTA2 variant.
      Two type A dissections at a diameter of <45 mm (1.77 inches) occurred in the peri- or postpartum period.
      • Regalado E.S.
      • Guo D.C.
      • Estrera A.L.
      • Buja L.M.
      • Milewicz D.M.
      Acute aortic dissections with pregnancy in women with ACTA2 mutations.
      We conclude that aortic diameter and z-scores solely are not reliable to estimate the risk for an aortic event and that hypertension and male sex should also be taken into account.
      PDA is described previously in individuals with an ACTA2-related vasculopathies, especially in individuals with the typical p.(Arg179His) variant causing multisystemic smooth muscle dysfunction syndrome.
      • Regalado E.S.
      • Mellor-Crummey L.
      • De Backer J.
      • et al.
      Clinical history and management recommendations of the smooth muscle dysfunction syndrome due to ACTA2 arginine 179 alterations.
      ,
      • Milewicz D.M.
      • Østergaard J.R.
      • Ala-Kokko L.M.
      • et al.
      De novo ACTA2 mutation causes a novel syndrome of multisystemic smooth muscle dysfunction.
      Beside the typical p.(Arg179His) variant in ACTA2, PDA is also described previously in 2 sisters with the ACTA2 variant p.(Asn117Lys).
      • Mc Glacken-Byrne A.B.
      • Prentice D.
      • Roshandel D.
      • et al.
      High-resolution iris and retinal imaging in multisystemic smooth muscle dysfunction syndrome due to a novel Asn117Lys substitution in ACTA2: a case report.
      However, we showed that it is can also be found in patients with other ACTA2 variants.
      In our cohort, 4% (2/49) of the individuals with different ACTA2 variants had a BAV, which is in line with the study of Guo et al.
      • Guo D.C.
      • Pannu H.
      • Tran-Fadulu V.
      • et al.
      Mutations in smooth muscle alpha-actin (ACTA2) lead to thoracic aortic aneurysms and dissections.
      Less than half of the individuals in this cohort had ocular abnormalities, mainly iris flocculi. As reported previously, most individuals with iris flocculi were heterozygote of the p.(Arg149Cys) ACTA2 variant.
      • Guo D.C.
      • Pannu H.
      • Tran-Fadulu V.
      • et al.
      Mutations in smooth muscle alpha-actin (ACTA2) lead to thoracic aortic aneurysms and dissections.
      ,
      • Chamney S.
      • McGimpsey S.
      • McConnell V.
      • Willoughby C.E.
      Iris Flocculi as an ocular marker of ACTA2 mutation in familial thoracic aortic aneurysms and dissections.
      The other 2 variants in our study associated with iris flocculi were novel, namely p.[Ala206Gly,Glu207_Ile269del] and p.(Thr162Asn).
      Tortuosity of the retinal vessels was observed in 1 patient and has also been reported previously, therefore, there might be a correlation with ACTA2-related vasculopathy.
      • Regalado E.S.
      • Mellor-Crummey L.
      • De Backer J.
      • et al.
      Clinical history and management recommendations of the smooth muscle dysfunction syndrome due to ACTA2 arginine 179 alterations.
      Keratoconus and retinal tear were identified once and have not been described thus far. It is uncertain whether these features are truly related to the ACTA2 variants. Previous studies have also reported aniridia and retinal detachment.
      • Regalado E.S.
      • Mellor-Crummey L.
      • De Backer J.
      • et al.
      Clinical history and management recommendations of the smooth muscle dysfunction syndrome due to ACTA2 arginine 179 alterations.
      Premature CAD, defined as age of onset less than 55 and 60 years in men and women, respectively, has been described in 20% of the individuals with a P/LP variant in ACTA2.
      • Guo D.C.
      • Papke C.L.
      • Tran-Fadulu V.
      • et al.
      Mutations in smooth muscle alpha-actin (ACTA2) cause coronary artery disease, stroke, and Moyamoya disease, along with thoracic aortic disease.
      The frequency of premature CAD in our cohort was lower (8%), which might be because of the low imaging rate of the coronary arteries. Both individuals with livedo reticularis carried the p.(Arg149Cys) ACTA2 variant and this is the only variant described so far in individuals with livedo reticularis.
      Only 1 individual had a dilated and tortuous course of the distal vertebral artery (2%), which is similar to the 2% of cerebral aneurysms reported by Guo et al.
      • Guo D.C.
      • Papke C.L.
      • Tran-Fadulu V.
      • et al.
      Mutations in smooth muscle alpha-actin (ACTA2) cause coronary artery disease, stroke, and Moyamoya disease, along with thoracic aortic disease.
      Other cerebral arteriopathy that have been described included abnormal straightening of the cerebral arteries, stenosis of the intra- and supraclinoid carotid artery, and the midbasilar artery.
      • Zhang A.
      • Jo A.
      • Grajewski K.
      • Kim J.
      Characteristic cerebrovascular findings associated with ACTA2 gene mutations.
      ,
      • Tschoe C.
      • Kim T.E.
      • Fargen K.M.
      • Wolfe S.Q.
      Cerebral arteriopathy in ACTA2 mutations: a spectrum of disease highlighted by a case of variable penetrance in two siblings.
      These abnormalities were not seen in our cohort.
      In 2018, a group of experts from the European reference network for rare vascular diseases had written a consensus statement for surveillance of individuals with a P/LP ACTA2 variant.
      • van de Laar I.M.B.H.
      • Arbustini E.
      • Loeys B.
      • et al.
      European reference network for rare vascular diseases (VASCERN) consensus statement for the screening and management of patients with pathogenic ACTA2 variants.
      This consensus statement recommends vascular surveillance of the asymptomatic adult heterozygotes of a P/LP ACTA2 variant using a 2-dimention–transthoracic echocardiography yearly and complete vascular imaging of thorax and abdomen every 2 to 5 years. Besides vascular surveillance, prophylactic treatment with beta-blocker from the age of 4 years is recommended.
      • van de Laar I.M.B.H.
      • Arbustini E.
      • Loeys B.
      • et al.
      European reference network for rare vascular diseases (VASCERN) consensus statement for the screening and management of patients with pathogenic ACTA2 variants.
      Despite the small cohort, our data give no reason to question the surveillance recommendations for asymptomatic individuals with a P/LP ACTA2 variant.

      Conclusion

      Heterozygous P ACTA2 variants show reduced penetrance for aortic disease, with hypertension and male sex as predisposing factors. Caregivers should be aware of aortic dissection, even in individuals with aortic diameters within the normal range. We conclude that mere aortic diameter and z-scores are not sufficient to predict the risk for an aortic dissection. Therefore, it is important to take into account other risk factors in individuals with an ACTA2 related vasculopathy like hypertension and male sex. We recommend aggressive treatment of hypertension. There seems to be no increased risk for complications with acute or prophylactic interventions.

      Data Availability

      Data are available upon request.

      Author Information

      Conceptualization: I.M.B.H.v.d.L., J.M.A.V., L.M.v.d.B.; Data Curation: I.M.B.H.v.d.L., J.M.A.V.; Formal Analysis: L.M.v.d.B.; Funding Acquisition: I.M.B.H.v.d.L.; Investigation: I.M.B.H.v.d.L., J.M.A.V., L.M.v.d.B., E.R.; Methodology: I.M.B.H.v.d.L., J.M.A.V., L.M.v.d.B.; Project Administration: I.M.B.H.v.d.L., J.M.A.V., L.M.v.d.B.; Resources: J.M.A.V., M.K., A.C.H., M.B., E.O., Y.H.-H., D.Q.C.M.B.-S., I.P.C.K., E.D., B.L.L., A.M., H.T.B., M.W.W., J.W.R.-H.; Supervision: I.M.B.H.v.d.L., J.M.A.V., J.W.R.-H.; Visualization: L.M.v.d.B., I.M.B.H.v.d.L.; Writing-original draft: L.M.v.d.B., J.M.A.V., I.M.B.H.v.d.L.; Writing-review and editing: J.M.A.V., J.A.B., M.K., A.C.H., M.B., E.O., Y.H.-H., D.Q.C.M.B.-S., E.R., I.P.C.K., E.D., M.W.W., B.L.L., H.J.M.V., A.M., J.W.R.-H., H.T.B., I.M.B.H.v.d.L., L.M.v.d.B.

      Ethics Declaration

      This study was approved by the Medical Ethics Committees of the Erasmus MC, University Medical Centre Rotterdam (MEC 2017-1162) and all affiliated centers. All patients provided written informed consent before their inclusion in this study.

      Conflict of Interest

      The authors declare no conflicts of interest.

      Acknowledgments

      We would like to thank all the research participants and the physicians and genetic counselors and investigators who have included individuals in this study and collected clinical data. This work was supported by the Dutch Heart Foundation (2014T007).

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