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Syndromic hereditary aortopathies are caused by mutations in the transforming growth factor-β-related genes.
Patients with Marfan syndrome exhibiting a FBN1 truncating variant carry an increased risk of aortic events.
There is a limitation to identify the underlying causative genes of non-syndromic juvenile aortopathies.
Genetic testing for hereditary thoracic aortic aneurysms and dissections should be performed with appropriate counseling.
Recent advances in DNA sequencing technology have identified several causative genes for hereditary thoracic aortic aneurysms and dissections (TAADs), including Marfan syndrome (MFS), Loeys–Dietz syndrome, vascular Ehlers–Danlos syndrome, and familial non-syndromic TAADs. Syndromic TAADs are typically caused by pathogenic variants in the transforming growth factor-β signal and extracellular matrix-related genes (e.g. FBN1, TGFBR1, TGFBR2, SMAD3, TGFB2, and COL3A1). On the other hand, approximately 20% of the non-syndromic hereditary TAADs result from altered components of the contractile apparatus of vascular smooth muscle cells, which are encoded by ACTA2, MYH11, MYLK, and PRKG1 genes; however, the remaining 80% cannot be explained by previously reported candidate genes. Moreover, the relationship between the genotype and phenotype of TAADs has extensively been reported to investigate better methods for risk stratification and further personalized treatment strategies. With regard to MFS-causing FBN1, recent reports have shown significantly increased risk of aortic events in patients carrying a truncating variant or a variant exhibiting a haploinsufficient-type effect, typically comprising nonsense or small insertions/deletions resulting in out-of-frame effects, compared to those carrying a variant with dominant negative-type effect, typically comprising missense variants. Therefore, cardiologists are required to have sufficient knowledge regarding the genetics of hereditary TAADs for providing the best clinical management, with an appropriate genetic counseling. In the current review, we present current advances in the genetics of hereditary TAADs and discuss the benefits and limitations with respect to the use of this genetic understanding in clinical settings.
Improved technology and decreased costs for DNA sequencing have identified the predisposition genes in hereditary thoracic aortic aneurysm and dissection (HTAAD), including those associated with Marfan syndrome (MFS), Loeys–Dietz syndrome (LDS), vascular Ehlers–Danlos syndrome (vEDS), and familial non-syndromic TAAD, and such genetic tests to guide precision medicine have been covered by health insurance in Japan since 2016. The ClinGen Aortopathy Expert Panel have classified 11 causative genes (FBN1, TGFBR1, TGFBR2, SMAD3, TGFB2, COL3A1, ACTA2, MYH11, MYLK, LOX, and PRKG1) for HTAAD as category A genes in 2018 (Table 1) [
]; this classification should trigger aortic imaging, medical therapy, and screening of family members for the variant with the goal of preventing acute aortic dissections, and thus cardiologists are required to have sufficient knowledge regarding the genetics of HTAAD for providing the best clinical management. In the current review, we present and discuss current advances in the genetics of HTAAD for its diagnosis and long-term management, with a focus on the category A genes.
Table 1Genes associated with familial thoracic aortic aneurysm
MFS is an autosomal dominant heritable disorder of the connective tissues with prominent involvement of skeletal, ocular, cardiovascular, and pulmonary organ systems. The estimated prevalence ranges from 1/5000 to 1/10,000, and approximately 75% cases of MFS have an affected parent and the remaining 25% of probands are sporadic [
]. Recent advances in surgical therapies for TAADs have improved life expectancy from approximately 30 years to >70 years; however, the comprehensive management strategies for preventing multiple organ system disorders remain inadequate to relieve lifetime anxiety and enhance the quality of life (QOL) of patients, despite the administration of β-blockers and angiotensin II receptor blockers, such as losartan [
], which place particular focus on the cardiovascular manifestations (aortic root aneurysm and/or dissection), ectopia lentis, and molecular genetic testing of FBN1. FBN1 spans about 230 kb of genomic DNA containing 66 exons (NM_000138.4), and >3000 different pathogenic variants have been identified and are distributed throughout the entire region of the gene [
]. In addition, we have identified 243 pathogenic and likely pathogenic variants (mutations) from 288 Japanese MFS families at our institute until February 2019. We have observed that missense mutations affecting/creating cysteine residues are the major variant type (85/243; 35.0%), followed by other missense (44/243; 18.1%), nonsense (44/243; 18.1%), in-frame and out-of-frame deletion/insertions (44/243; 18.1%), and splice (26/243; 10.7%) mutations, which is consistent with data previously reported [
The encoded fibrillin-1 is a major component of elastic fibers that are widely expressed in structural elements of the affected organs and tissues, and MFS is traditionally considered to result from the structural weakness of the connective tissue. However, recent reports on molecular mechanisms of MFS have indicated that fibrillin-1 interacts with latent transforming growth factor-β (TGF-β) binding protein-1 (LTBP) to regulate TGF-β activity (Fig. 1A ), and therefore defective fibrillin-1 results in the structural weakness and excessive activity of TGF-β signaling—both of which contribute to the complicated pathogenesis [
]. Fibrillin-1 contains 7 TGF-β binding protein-like (TB) domains and 47 epidermal growth factor (EGF)-like domains (Fig. 1B), which include 8 and 6 cysteine residues that are involved in 4 and 3 intramodule disulfide bond formation, respectively. Of the 47 EGF-like domains, 43 contain a consensus sequence for calcium binding (cb-EGF) that is typically present as multiple tandem repeats and play a crucial role in microfibril stability and assembly [
Although there are no obvious mutational hot spots and the precise molecular mechanism for how mutations affect fibrillin-1 structure and function remains largely elusive, the relationships between the location or type of FBN1 mutations and phenotypes have been extensively reported to search for better methods for risk stratification and personalized treatment strategies [
]. It has long been known that a higher probability of ectopia lentis is found in patients with a missense variant affecting a cysteine residue particularly in the first 16 exons, and exons 25–33 including a central stretch of cb-EGF domains are recognized as a critical region for severe form of neonatal MFS, which is characterized by severe mitral and/or tricuspid valvular insufficiency and pulmonary emphysema. In addition, recent reports have shown significantly increased risk of aortic events in patients carrying a truncating variant or a variant with a haploinsufficient (HI)-type effect, typically comprising nonsense and splice site variant, or small insertions/deletions leading to out-of-frame effects, compared to patients carrying a variant with dominant negative (DN)-type effect, typically comprising missense variants or small insertions/deletions leading to in-frame effects (Fig. 2) [
To elucidate the relationship between FBN1 genotype and severe aortopathy (aortic root replacement, type A dissections, and related death) in Japanese patients with MFS, we have recently evaluated 248 patients with pathogenic or likely pathogenic FBN1 variants [
]. Only FBN1 variants fulfilling the 2015 American College of Medical Genetics and Genomics-Association for Molecular Pathology criteria for pathogenicity (≥class 4) were included. Variants were simply subcategorized into 2 groups (HI and DN) according to the predicted effects on protein structure and function; however, most splice site variants were classified into DN variants in this study, because most exons in FBN1 have lengths that are multiples of 3 bp and the defective proteins presumably exert in-frame exon skipping effects. Prophylactic aortic root replacement had been recommended for those having an aortic diameter ≥45 mm at our institute.
The main results from our Japanese patients were consistent with previous data from non-Japanese patients that show HI patients had a 2.1-fold higher risk of severe aortic events compared with DN patients, and male patients had an increased risk: the median event-free survival period from birth for men and women was 31.0 and 41.0 years, respectively, in HI group and 41.0 and 55.0 years, respectively, in DN group [
]. The increased risk in men is attributed not only to the absolute threshold for aortic surgery independent of body size and the larger overall body surface area of men, but also to rapid growth in men. In addition, we newly identified an early-onset genetic subgroup within the slow-onset DN group, showing that patients with variants affecting or creating Cysteine residues and in-frame Deletion (DN-CD) variants in the central tandem cb-EGF domains (exons 26–37, c.3083–4582 and 44–50, c.5297–6163) were as deleterious as HI patients and were at a 6.3-fold higher risk than those with other types of DN variants (DN-nonCD) (Fig. 3) [
]. Because the underlying mechanisms are unknown and variability in phenotypes has been reported not only across families with the same FBN1 genotype but also within families, MFS patients should not be monitored based on genotype alone. However, we believe that our simple genetic classification would provide valuable directives and perspectives to patients and relatives with tremendous amount of anxiety during their long-term follow-up period. Further investigations are expected to reveal the relationship between FBN1 genotype and other manifestations influencing QOL, such as kyphoscoliosis and myocardial function as well as potential differences in drug response.
LDS is a recently established autosomal dominant heritable MFS-like syndrome, caused by pathogenic variants in TGF-β signal-related genes, and LDS patients were classified according to the mutated genes in the 2014 revised nosology for LDS diagnosis: TGFBR1 (LDS1), TGFBR2 (LDS2), SMAD3 (LDS3), and TGFB2 (LDS4) [
]. TGFB3 (LDS5) and SMAD2 (LDS6) have been also recently reported to be causing genes for MFS-like syndromic HDAAD, but are still assigned to category “uncertain” for the clinical validity in 2018 (Table 1) [
], pending additional evidence showing the severity of associated aortic disease and risk of progression. Estimated incidence of LDS is approximately 5%–10% among patients with suspected MFS, and the frequency of mutation in the TGFBR2 seems to be more frequent (55%–60%) than other gene mutations (TGFBR1, 20%–25%; SMAD3, 5%–10%, TGFB2, 5%–10%; TGFB3, 1%–5%; and SMAD2, 1%–5%) [
Vascular and skeletal features in LDS demonstrate overlap with those of MFS, and 20% LDS patients have systemic score of ≥7 points of the 2010 revised Ghent criteria for MFS (Table 2). However, LDS patients do not show ectopia lentis and the majority do not have prominent overgrowth of the long bones (tall height and long arms and legs), and approximately 10%–20% of patients have congenital heart defects, such as patent ductus arteriosus (PDA), atrial septal defect, bicuspid aortic valve (BAV), and ventricular septal defect (VSD) [
]. Further characteristically, LDS patients have a triad of clinical features: hypertelorism (widely spaced eyes), cleft palate or bifid (split) uvula, and aortic/arterial tortuosity and aneurysms (Fig. 4), and other LDS-specific features can include cervical spine malformation and/or instability, translucent skin with easy bruising, and dystrophic scars. These indicate that TGF-β signaling pathways play crucial roles in the development and maintenance of various tissues, including arteries and craniofacial growth and patterning. Pathogenic variants in LDS are predicted to lead to loss of protein function; however, aortic tissues from affected patients and LDS knock-in mice showed overactivity of TGF-β signaling cascades, and the molecular mechanisms for the TGF-β paradox have been actively investigated [
Pathogenic variants in TGFBR1 and TGFBR2 are mostly missense and detected within the serine/threonine kinase (STK)-encoding regions of the receptors. Although TGFBR2 forms heteromeric complex with TGFBR1 and cooperatively initiates signaling after binding TGF-β ligands, clinical features associated with a pathogenic variant in TGFBR1 slightly differ from that in TGFBR2 [
]; to date, patients with LDS1 and LDS2 had been recognized to rapidly develop progressive aortic/arterial aneurysms, resulting in ruptures at an early age and at smaller dimensions, and surgical repair of the aortic root has been recommended earlier than MFS, potentially at a diameter of ≥40 mm in Japan, and indeed even children <10 years of age have a higher risk for developing aortic dissections (Fig. 4B) [
]. However, recent international registry data gathered by the Montalcino Aortic Consortium (MAC 2016) revealed that the survival of patients with a TGFBR1 or a TGFBR2 pathogenic variant is much better than initially reported [
]. In addition, TGFBR1 males have a greater aortic risk than females, whereas there is no similar sex effect among TGFBR2 patients, and TGFBR2 females are prone to develop type A aortic dissection at a diameter ≤45 mm. Furthermore, aortic tortuosity, hypertelorism, and translucent skin are associated with an increased aortic dissection risk, suggesting that syndromic patients with these LDS phenotypic features should be managed more aggressively than patients lacking them [
]; however, the MAC recommends prophylactic surgery when the diameter reaches 45 mm, and the threshold could be lowered toward 40 mm in TGFBR2 females with low body surface area and presenting with extra-aortic features [
With respect to LDS3–6, the reported number of patients with SMAD3, TGFB2, TGFB3, or SMAD2 pathogenic variants may be inadequate to draw any conclusions on the phenotypic spectrum. In 2018, a multi-country survey on SMAD2/3 and TGFB2/3 variants summarized and compared the clinical features of LDS3–6 patients with 45 new pathogenic variants as well as 74 previously reported ones [
]: The aortic phenotype in LDS3 patients is similar to that of LDS1/2 patients and LDS4/5 represents mild cardiovascular features. Accordantly, the recommended threshold diameter for prophylactic surgery in LDS3 and LDS4 is 40 and 50 mm in the 2014 revised nosology, respectively [
In MAC study, there seem not to be increased risks of the occurrence of uterine rupture and pregnancy-related aortic dissections in LDS1/2 compared to MFS patients that were initially emphasized in 2006 [
]. Further investigations on relationship between LDS genotype and phenotype are needed to detect high-risk subgroups warranting improved risk stratification and management.
Vascular Ehlers–Danlos syndrome
EDS is a clinically and genetically heterogeneous group of hereditary connective tissue disorders characterized by skin hyperextensibility, joint hypermobility, and tissue fragility, which is caused by pathogenic variants of genes encoding collagen or collagen-modifying enzymes [
], but vEDS (previously known as EDS type IV) is considered the most serious form of EDS owing to the possibility of arterial dissection and rupture, intestinal perforation, or uterine rupture (Table 3). Pepin et al. reviewed clinical records in 1231 individuals with vEDS (630 index cases and 601 relatives), the median survival is 46 ± 1.8 years for males and 54 ± 2.5 years for females, with an increased number of deaths in young males [
]. vEDS patients often present with easy bruising, characteristic facial appearance, and thin skin with increased venous visibility; however, it is often difficult to make a diagnosis until serious complications occur.
Table 3Diagnostic criteria for vascular Ehlers–Danlos syndrome
(1) Family history of vEDS with documented causative variant in COL3A1
(2) Arterial rupture at a young age
(3) Spontaneous sigmoid colon perforation in the absence of known diverticular disease or other bowel pathology
(4) Uterine rupture during the third trimester in the absence of previous C-section and/or severe peripartum perineum tears
(5) Carotid-cavernous sinus fistula formation in the absence of trauma
(1) Bruising unrelated to identified trauma and/or in unusual sites such as cheeks and back
(2) Thin, translucent skin with increased venous visibility
(3) Characteristic facial appearance
(4) Spontaneous pneumothorax
(6) Talipes equinovarus
(7) Congenital hip dislocation
(8) Hypermobility of small joints
(9) Tendon and muscle rupture
(11) Gingival recession and gingival fragility
(12) Early-onset varicose veins (under age 30 and nulliparous if female)
Minimal criteria suggestive for vEDS:
- A family history of the disorder, arterial rupture, or dissection in individuals <40 years of age, unexplained sigmoid colon rupture, or spontaneous pneumothorax in the presence of other features consistent with vEDS should all lead to diagnostic studies to determine if the individual has vEDS.
- Heterozygous mutation in the COL3A1 gene
- Three heterozygous COL1A1 mutations leading to a arginine-to-cysteine substitution: c.934C>T, p.Arg312Cys; c.1720C>T, p.Arg574Cys and c.3277C>T, p.Arg1093Cys
]. Three identical pro-alpha1(III) chains, consisting of a central collagenous domain characterized by the repeated amino acid motif [Glycine (Gly)-Xaa-Yaa]343, where Xaa and Yaa can be any amino acid, and two flanking N- and C-terminal noncollagenous domains, are assembled into type III pro-collagen homotrimers (Fig. 5). Noncollagenous domains are proteolytically removed and triple helices formed associate laterally to form collagen fibrils with a characteristic banded pattern.
More than 700 unique COL3A1 pathogenic variants have been identified and approximately 50% of cases are sporadic. The relationship of COL3A1 genotype to vascular phenotype has been also analyzed [
]: The majority (∼2/3) are missense mutations affecting the glycine residue of Gly-Xaa-Yaa triplets. The mutated residues were mostly either valine (Val), glutamate (Glu), or aspartate (Asp), and this selection bias is positively correlated to the triple helix destabilizing effects [
]. Most of the remaining pathogenic variants are located within potential splice-sites leading to an in-frame exon skipping and generation of a shorted translated product. These 2 genotypes are at high risks for arterial complications and survival. In contrast, patients with nonsense and small insertion/deletion variants leading to HI-type effects, and non-glycine sequence and N- and C-terminal missense variants, are reported to have milder clinical features and/or present at older age. These might indicate that DN-type variants profoundly disrupting and/or destabilizing the collagen triple helical structure are associated with a more severe phenotype, and it is also important to suspect that non-syndromic-appearing patients with milder arterial features may have the milder types of COL3A1 variants, even though some variants will require biochemical studies to reveal their causal role on type III collagen alteration of assembly and/or production [
] as causal for vEDS that are associated with rupture of medium-sized arteries in adult age as well as classic EDS presentation.
Familial non-syndromic thoracic aortic aneurysm and dissection
Familial non-syndromic TAADs could be caused by pathogenic variants in genes coding proteins with functionality specific to the aorta, and the defects cause only TAADs with few outward physical manifestations; surgical repair is typically recommended when the ascending aortic size is 4.5–5.0 cm. The ClinGen Aortopathy Expert Panel classified 5 causative genes for HTAAD as category A2 genes primarily causing isolated TAAD [
]: ACTA2, MYH11, MYLK, and PRKG1 encode components of the contractile apparatus of the vascular smooth muscle cells (SMC), and LOX encodes an extracellular copper enzyme that catalyzes the cross-linking of collagens and elastin. However, the genetic etiology is substantially heterogeneous and approximately 80% of all cases of familial non-syndromic TAADs cannot be explained by pathogenic variants in any of these genes [
]. The natural history and clinical events of non-syndromic TAADs has not been well established; therefore, the diagnosis of familial non-syndromic TAADs is difficult. The management of family members should be carefully and appropriately performed in compliance with ethical guidelines for genetic testing.
ACTA2 is the major responsible gene for familial non-syndromic TAADs (14%–21%) and encodes aortic α-smooth muscle actin protein [
], a transcriptional target of the TGF-β signaling. All mutations identified to date are either missense or in-frame insertion-deletion (indel) mutations with DN-type effect, and currently, there is no evidence to support that the HI variants predispose to TAADs [
]. Although the penetrance is approximately 50%, patients frequently develop other vascular features and/or functional disorders of SMCs, such as cerebral aneurysm, neurovascular malformation resembling moyamoya disease, coronary artery disease, PDA, livedo reticularis, and iris floccule [
Other causative genes contribute remarkably less to the genetic etiology of non-syndromic TAADs (≤1%). MYH11 encodes SM myosin heavy chain (Myosin 11), and a considerable number of genetic variants of unknown significance (UVS) are found throughout the gene; however, most pathogenic variants responsible for TAADs are either missense or in-frame indel mutations within the coiled-coil domain and interfere with protein interactions [
]. Although the penetrance is incomplete, most pathogenic variants in MYH11 have been found in patients with TAAD exhibiting PDA.
SMC contractile forces are generated by cyclic interactions between SM myosin heads and the actin filaments and regulated by the reversible phosphorylation of myosin light chain (MLC); Ca2+/calmodulin (CaM)-dependent MLC kinase (MYLK) phosphorylates MLC and increases myosin ATPase activity, creating actin–myosin cross-bridges for contraction, whereas MLC phosphatase (MLCP) dephosphorylates MLC to inhibit the cross-bridge formation resulting in the SMC relaxation, and the activity is increased by cGMP-dependent protein kinase-1 (PKG-1) (encoded by PRKG1). Consequently, mutations in MYLK and PRKG1 have been reported to cause TAADs [
], by rendering the aorta less competent to withstand biomechanical forces from pulsatile blood flow.
MYLK produces 3 isoforms and the only 130-kDa short form (amino acids 923–1914) is expressed in human aorta, which contains the catalytic and CaM domain required for activation and kinase activity, and heterozygous MYLK mutations cause either haploinsufficiency or destroy the CaM binding domain [
], and thus the management of their relatives should be particularly careful. In the PRKG1 gene, a recurrent gain-of-function mutation (c.530G>A; p.Arg177Gln) causes TAADs with complete penetrance. The p.Arg177Gln mutation disrupts binding to the high-affinity cGMP binding site; however, the altered PKG-1 is constitutively active even in the absence of cGMP and hypertension is present in several patients [
LOX encodes lysyl oxidase (LOX), also known as protein-lysine 6-oxidase, plays a critical role in extracellular matrix maturation by cross-linking collagen and elastin. The heterozygous loss-of-function mutation, particularly variants that disrupt the catalytic activity or lead to haploinsufficiency, predisposes to TAADs involving both aortic root and ascending aorta [
]. Patients exhibit some overlapping syndromic features, such as pectus deformities and striae, but do not fulfill the diagnostic criteria for MFS, and mutation carriers frequently present with BAV (∼15%) [
]. Recent progress in sequencing technology would facilitate further discovery of new causative genes, and targeted multi-gene panel testing for HTAADs makes it possible to screen multiple candidate genes simultaneously in a clinical setting, including category non-A genes, such as SKI for Shprintzen–Goldberg syndrome (SGS) and FBN2 for congenital contractural arachnodactyly (also known as Beals syndrome). These can facilitate an easier and more efficient diagnosis; however, doctors and/or genetic counselors must remember to explain the benefits, risks, and limitations in detail before testing. Genetic testing can reveal information about family members, which can still result in genetic discrimination in employment or insurance. In addition, the negative test result of the proband for some known TAADs-causing genes does not suggest that family members do not have an increased risk of developing TAADs in the future. Furthermore, at the present, genetic testing does not provide adequate information on how the disorder will progress over time. Further genetic investigations are required to put precision medicine into practice, with the appropriate genetic counseling.
This work was supported by a Grant-in-Aid for Research on Rare and Intractable Diseases from the Japan Agency for Medical Research and Development .
Conflict of interest
The authors declare that there is no conflict of interest.
We are grateful to the members of Marfan Clinic at the University of Tokyo Hospital for valuable discussion and contributions.
Clinical validity of genes for heritable thoracic aortic aneurysm and dissection.