Systematic Review of Studies That Have Evaluated Screening Tests in Relatives of Patients Affected by Nonsyndromic Thoracic Aortic Disease

Background Nonsyndromic thoracic aortic diseases (NS‐TADs) are often silent entities until they present as life‐threatening emergencies. Despite familial inheritance being common, screening is not the current standard of care in NS‐TADs. We sought to determine the incidence of aortic diseases, the predictive accuracy of available screening tests, and the effectiveness of screening programs in relatives of patients affected by NS‐TADs. Methods and Results A systematic literature search on PubMed/MEDLINE, Embase, and the Cochrane Library was conducted from inception to the end of December 2017. The search was supplemented with the Online Mendelian Inheritance in Man database. A total of 53 studies were included, and a total of 2696 NS‐TAD relatives were screened. Screening was genetic in 49% of studies, followed by imaging techniques in 11% and a combination of the 2 in 40%. Newly affected individuals were identified in 33%, 24%, and 15% of first‐, second‐, and third‐degree relatives, respectively. Familial NS‐TADs were primarily attributed to single‐gene mutations, expressed in an autosomal dominant pattern with incomplete penetrance. Specific gene mutations were observed in 25% of the screened families. Disease subtype and genetic mutations stratified patients with respect to age of presentation, aneurysmal location, and aortic diameter before dissection. Relatives of patients with sporadic NS‐TADs were also found to be affected. No studies evaluated the predictive accuracy of imaging or genetic screening tests, or the clinical or cost‐effectiveness of an NS‐TAD screening program. Conclusions First‐ and second‐degree relatives of patients affected by both familial and sporadic NS‐TADs may benefit from personalized screening programs.

D iseases of the thoracic aorta are increasing in prevalence, accounting for 1% to 2% of all deaths in Western countries. [1][2][3][4][5] In the United States, diseases of the aorta account for more than 40 000 deaths per year. 1,4 Thoracic aortic diseases (TADs) are often silent entities with a mortality of almost 80% when presenting as life-threatening emergencies. 3,6 Therefore, early diagnosis and treatment are likely to improve long-term survival. TADs may be syndromic, associated with disorders involving other organs such as Marfan syndrome, or more commonly nonsyndromic, with manifestations restricted to the thoracic aorta. 4,5 Nonsyndromic TADs (NS-TADs) may be familial, characterized by the presence of a family history and an autosomal dominant inheritance, or sporadic. [4][5][6][7] Unlike syndromic TADs, NS-TADs are not evident from external physical features and abnormalities of other organ systems and are characterized by silent aneurysm formation and dissection. 4,5 Screening of first-degree relatives (FDRs) of patients affected by NS-TAD is therefore recommended for early detection and treatment of asymptomatic disease. 4,5 However, existing guidelines are based predominantly on the consensus of expert opinion, rather than high-quality evidence, and the testing modality, frequency, and extent (FDRs versus second-degree relatives [SDRs]) of screening are not defined. 4,5 As a consequence, there is widespread variation in the screening of family members of patients with NS-TADs. To address this area of

Participants
We included studies considering imaging and/or genetic screening tests in probands affected by diseases of the thoracic aorta (aneurysms and/or acute aortic syndrome), and their FDRs, SDRs, and third-degree relatives (TDRs), with no restriction on ethnicity or age.

Target Condition
The target condition was disease of the thoracic aorta (aneurysm and/or acute aortic syndrome) defined by the international guidelines on diagnosis and management of patients with TAD. 4,5 Only NS-TAD forms were considered in the present review; syndromic TADs or other forme fruste of syndromic TAD related to the transforming growth factor b pathway were excluded. Familial NS-TAD forms were defined as those occurring in families with ≥2 members with a known TAD, but without a clinical diagnosis or history of a syndromic TAD or any other connective tissue disease. 7 Sporadic TADs were defined as those occurring in patients apparently without a family history of TAD or evidence of syndromic TAD. 4,5,7

Index Tests
For the purposes of the review, we included studies that phenotyped participants using the following imaging tests: transthoracic echocardiogram (TTE)/transoesophageal echocardiogram, computed tomography (CT), or magnetic resonance imaging (MRI) of the thoracic aorta, and genetic screening, individually or in combination with the acknowledgement that sensitivities and specificities of CT (100% and 100%, respectively) and MRI (95-100%) are higher when compared with those of transoesophageal echocardiogram and TTE (74-100% and 71-91%, respectively). [11][12][13][14][15] In some studies, surgery for TAD, postmortem examination, or sudden death were used to assess the aortic phenotype. Molecular genetic testing approaches included a combination of genetargeted testing (multigene panel or single gene testing) and whole exome of genome sequencing. [16][17][18] Study Selection, Data Collection, and Extraction Two investigators (G.M. and R.D.) independently reviewed titles, abstracts, and full-text articles against the specified inclusion criteria for studies regarding screening of relatives Clinical Perspective What Is New?
• Imaging and/or genetic screening is not the current standard of care in relatives of patients affected by nonsyndromic thoracic aortic diseases (NS-TADs). • Genetic and/or imaging screening of relatives of patients affected by NS-TAD can detect more than 30% of patients newly affected by thoracic aortic diseases.
What Are the Clinical Implications?
• Routine imaging and genetic testing of relatives of patients affected by nonsyndromic aortopathies should be encouraged. • The evidence suggests that screening of firstand seconddegree relatives of patients affected by familial NS-TAD and first-degree relatives of those affected by sporadic NS-TADs will result in significant numbers of patients with otherwise undiagnosed disease. • Personalized screening programs determined by the subtype of NS-TAD and its related genetic mutation have the potential to benefit these patients.
of patients with NS-TADs. Discrepancies were resolved through consensus and consultation with a third investigator (G.J.M.). One reviewer extracted key data from the included studies using a standard dedicated pro forma; a second reviewer checked the collected data for completeness and accuracy. The Tables report full details on study design and quality, setting and population, details, and results of screening. Key study characteristics include details of the patient population (NS-TAD form, ethnicity, family identification), participants undergoing screening (relatives eligible for screening; family pedigree; total number of screened relatives; numbers of FDRs, SDRs, and TDRs), TAD characteristics (new diagnosis of aortic disease, number/rate of newly diagnosed thoracic aortic aneurysms and/or dissection, rate of unexplained sudden death, age and aortic diameters at dissection, sex preponderance, and aortic disease penetrance), additional concomitant phenotype/clinical features (types and rates), and type of adopted screening modality (imaging and genetic test used, validation processes). The definitions of the extracted variables are fully reported in Data S1.

Quality Assessment, Data Synthesis, and Analysis
Two investigators (G.M., R.D.) independently appraised all articles that met inclusion criteria. Study quality was assessed using the Newcastle-Ottawa Scale and the US Preventive Services Task Force. 19,20 The Cochrane Risk of Bias tool was also used to evaluate the methodological quality of all included studies. 21 Because of the observational nature of the studies and their clinical heterogeneity, the analyses were largely descriptive, and a narrative and tabular synthesis of all included studies is provided. Inclusion and exclusion criteria for qualitative/quantitative analyses are summarized according to the PICOS (population, intervention, comparator, outcomes, and study design) approach (Table S2). Subgroup analysis considering type of NS-TAD form, aortic disease (aneurysm and/or dissection), genetic mutation, and screening modality was also conducted. Categorical variables are reported as number and percentage, and continuous variables are reported as mean and SD or median and range, according to distribution. Analyses were performed with SPSS version 24.0 (IBM).

Description of Studies and Quality Assessment
Of the 12 897 records identified, 53 studies were included in the systematic review, comprising a total of 2696 screened relatives. The studies were published between 1985 and 2017 ( Figure S1).  Regions of origin included North America (28 studies), Europe (17 studies), Asia (5 studies), and Australia (3 studies) ( Table 1). No randomized trials were identified, and only 1 large cross-sectional study was conducted including 581 at-risk relatives. 58 Study characteristics and collected outcomes are summarized in Tables S3 through S8 and study  quality assessment in Table S9.

Target Condition
Four main groups of familial NS-TADs were identified: (1) those characterized by the presence of both aneurysms and dissections in the family pedigree (familial thoracic aortic aneurysm and dissection; 44 studies); (2) those characterized by aneurysmal disease only (familial thoracic aortic aneurysm; 2 studies); (3) those characterized by aortic dissection only (familial thoracic aortic dissection; 3 studies); and (4) thoracic aortic aneurysm forms associated with the presence of bicuspid aortic valve (4 studies). Among the familial thoracic aortic aneurysm and dissection forms, 3 additional subgroups were discovered based on the concomitant presence of patent ductus arteriosus (n=4), intracranial aneurysms (n=2), or peripheral arterial aneurysms (n=2) ( Table 1).

Index Tests
Screening for TAD was performed using 2-dimensional TTE in 27 (51%) studies, of which 15 (28%) employed 2-dimensional TTE alone and the remaining 8 (15%) used 2-dimensional TTE in association with CT and/or MRI. In 5 (9%) studies only, imaging screening included the simultaneous employment of 2-dimensional TTE, CT, and MRI. In a further 26 (49%) studies, aortic phenotype (presence of an aortic aneurysm and/or dissection) was defined by reported clinical events including acute aortic syndrome, diagnosis made during routine diagnostic clinical care, or postmortem examination. The aortic diameter cutoff used for defining a critical dilation of the aorta varied among studies as the aortic site where the measurements were made (Table S7).
No study reported the sensitivity, specificity, or other measures of diagnostic accuracy for the index tests. One study reported 10-year longitudinal follow-up for relatives of patients with NS-TAD. 59 In this study, relatives with evidence of aortic dilatation were offered annual follow-up imaging with prescription of b-blockers or angiotensin receptor blockers at maximal tolerated doses. Relatives with no evidence of aortic dilatation (unaffected) were subjected to clinical review every 3 years. In the affected relatives (n=114) with serial aortic measurements over 4.5AE4.4 years, a mean rate of increase in the aortic diameter of 0.56AE0.76 mm per year was observed. No difference in the rate of aortic dilatation was observed between males and females or in patients receiving    intracranial aneurysm; IMAG, imaging; n/a, not available; NS-TAD, nonsyndromic thoracic aortic disease; n/c, not computable; pAA, peripheral artery aneurysm; PDA, patent ductus arteriosus; SDRs, second-degree relatives; TAA, thoracic aortic aneurysm; TAAD, thoracic aortic aneurysm and/or dissection; TDRs, third-degree relatives. *Study performed at University of Texas.
b-blockers or angiotensin receptor blockers. No correlation with the age at diagnosis, the initial aortic diameter, and the systolic or diastolic blood pressure was documented. During 10-year follow-up, 9% of newly diagnosed relatives were affected by an aortic dissection, and 18% underwent elective aortic surgery. Six relatives (of 368) originally diagnosed as unaffected (initial aortic diameter with a Z score <2) experienced a subsequent aortic dissection. 59

Results of Imaging Tests
A total of 1039 families underwent screening for NS-TAD, with a median number of patients in each family pedigree of 48 (study range: 6-270) ( Table S3). The proportion of potential eligible patients per family was 73% (study range: 50-100%), while the rate of relatives effectively screened was 54% (study range: 5-100%) ( Table 2 and Table S4). FDRs, SDRs, and TDRs were variably screened throughout the studies. Twelve percent of FDRs, 24% of SDRs, and 18% of TDRs were not available for screening ( Figure 1). A total of 893 FDRs, 695 SDRs, and 670 TDRs were identified in the family pedigrees of the included studies (Table S5). Of these, a total of 910 newly affected relatives were detected, with an average among studies of 22 newly diagnosed individuals. The percentage of newly diagnosed relatives was 23% (study range: 6-56%). Newly diagnosed individuals were male in 67% of the cases (study range: 20-100%). Sudden unexplained deaths were reported in 2% of the cases (study range: 0-9%). Detailed data about rates of newly affected and screened FDRs, SDRs, and TDRs are depicted in Figure 1.
The type of aortic diseases (aneurysm and dissection rates), male preponderance rate, and age at dissection are summarized in Table 2 and Table S4. Only 1 study screened the relatives of 53 probands identified as affected by a sporadic NS-TAD form, identifying 83 of 321 newly affected relatives. 59

Results of Genetic Tests
The techniques used in the genetic screening, the identified genes, and genetic mutations are listed in Table S8. Genetic screening was employed as the sole screening modality in 26 (49%) studies and in combination with imaging modalities in 21 (40%). A total of 14 known genes were identified as a causative mutation for NS-TADs, while 3 mapped loci without an identified gene were also found (Table 3 and Table S11). Single-gene testing was used in 24 (45%) studies, comprehensive genomic sequencing in 14 (26%), and a combination of the 2 approaches in 7 (13%), respectively ( Figure 2, Tables S8 and S10).
The inheritance mode was essentially autosomal dominant (Table 1). Forty-one (79%) studies reported on the penetrance of the NS-TAD. Penetrance varied in relationship to the NS-TAD form, with an average of 67% (study range: 20-100%) and was lower in females ( Table 2). The age at dissection varied according to the underlying NS-TAD form, with a mean age of presentation of 32 years for the familial thoracic aortic aneurysm and dissection forms associated with the mutations of the PRKG1 gene and of 54 years for those associated with the mutation of the MYLK gene ( Figure 2 and Table 3). Ascending aortic diameters at the time of acute dissection were not reported for most of the individuals. Where this was reported, individuals affected by NS-TADs showed stratification of the diameter of the thoracic aorta (aortic root, mid ascending, or descending aorta) at dissection and in the risk of progression to dissection by genetic mutation: from <4.5 cm for FBN1, FOXE2, MILK, PRKG1, SMAD3, TGFB2, TGFBR1, and TGFBR2, to >5.5 cm for ACTA2, LOX, and MYH11, respectively ( Figure 2, Table S10). The identified NS-TAD forms presented specific characteristics based on the causative genetic mutation (Tables 1 and 3, Table S10, and Figure S2).

Extra-Aortic Manifestations of NS-TAD
Concomitant cardiovascular diseases were diagnosed in 11% of the relatives undergoing screening, while concomitant physical abnormalities were observed in 18% of the cases. Full details of all described external physical features and abnormalities of other organ systems are reported in Table S6.

Resource Use and Cost-Effectiveness
No information about resource use and the cost-effectiveness of screening program in relatives of patients with NS-TAD was reported in any of the identified studies. No studies address the psychological effect of screening in patients and their relative or its impact on quality of life of these families.

Main Findings
The present study has identified an area of unmet clinical need with respect to screening of relatives of patients with NS-TAD: familial NS-TADs occur more frequently than previously recognized, affecting %30% of relatives with a male predominance (3:1). These are primarily inherited as single gene mutations, expressed in an autosomal dominant pattern with incomplete penetrance, which demonstrate variable expression with respect to age of presentation, sex, aneurysmal location, and aortic diameter before dissection. The risk of acute aortic syndrome is determined by the underlying   Table 2. n/a n/a n/a indicates not available.
*Percentage calculated in the family pedigree (as per protocol).
genetic mutation and this risk extends not only to FDRs but also to SDRs and TDRs of patients affected by NS-TADs. There is an overlap between nonsyndromic and syndromic TADs for some genetic mutations, as well as concomitant cardiovascular pathology in over 10% of screened patients. The review also identified knowledge gaps with respect to the predictive accuracy of commonly used screening tests across NS-TAD populations, the optimal structure and extent of a screening program across families, and the effectiveness of a screening program with respect to clinical outcomes or cost.

Clinical Implications
Nonsyndromic aortopathies have poor prognosis if untreated and the lack of relevant physical features precludes identification based on a clinical characteristics alone. 7,75 As a consequence, NS-TADs are asymptomatic, alerting clinicians to the underlining aortopathy only when sudden death or an acute aortic dissection occurs. 7,19,72,75 This review indicates that routine screening and surveillance programs in relatives of patients affected by NS-TADs, similar to those of syndromic TAD, are likely to identify significant numbers of patients with asymptomatic NS-TAD. 4,5,76,77 The overlap in genetic mutations between NS-TAD and syndromic TAD identified in the review further support this assertion. It follows that diagnosis, surveillance, and treatment of NS-TADs before clinical presentation, as is the standard of care for syndromic TAD, is likely to reduce premature deaths. The findings of this article also indicate that current guidelines which recommend treatment based predominantly on the aortic diameter are likely to result in the undertreatment of NS-TADs. 4,5 Specifically, subtypes of NS-TADs attributed to specific genetic mutations may progress to aortic dissection without aneurysm formation. 78 Here, the treatment of affected relatives stratified by NS-TAD subtype and genetic abnormality are likely to result in further clinical benefits ( Figure 2). In addition to defining an area of unmet need, the review has identified important knowledge gaps with respect to screening. Specifically, the diagnostic accuracy of existing screening tests, the optimal screening program, and the clinical, societal, or economic benefits of such a screening program in the relatives of patients with sporadic or familial NS-TAD are unclear. Current guidelines for the diagnosis and treatment of aortic diseases do not specify the details of what screening tests should be used (Table S11). 4,5,77 The 2014 European Society of Cardiology guidelines recommend investigating FDRs by genetic counseling for family investigation and molecular testing, with a 5-year interval screening until diagnosis (clinical or molecular) is established or ruled out (class I, level of evidence C). 5 The corresponding 2010 American guidelines suggest aortic imaging screening for FDRs along with counseling and testing whether a specific mutant gene (FBN1, TGFBR1, TGFBR2, COL3A1, ACTA2, MYH11) is identified in the affected probands (class I, level of evidence C). 4 These recommendations are based on opinion of the experts and small group studies only. 4,5 Importantly, specific testing schedules, the requirement for screening of SDRs and TDRs, the need for sequencing of other lesscommon mutant genes, the optimal screening interval and modality, or the need to investigate the entire arterial tree     other than the thoracic aorta are not specified. 4,5 The results of the current study suggest that FDRs, SDRs, and possibly TDRs should be offered screening for TAD. Clarification of the family history regarding the location of the aortic disease, the specific nature of "sudden deaths," or the presence of other concomitant cardiovascular disorders during clinical examination should represent the initial step of screening. 75 Our results also suggest that genetic testing and cardiac imaging with at least TTE should be offered to all FDRs and SDRs of patients with suspected NS-TADs. Mutation carriers should undergo further imaging (MRI or CT scan), focusing on thoracic aorta and/or other arterial trees based on the causative gene mutation.  For example, ACTA2-mutation carriers should be monitored for coronary artery disease and occlusive cerebrovascular disease, in addition to the currently recommended routine imaging tests. 32 We suggest that complete aortic imaging at initial diagnosis and at 6 months for patients with a confirmed genetic aortopathy (eg, FBN1, FOXE3, MFAP5, MYLK, PRKG1, SMAD3, TGFB2, TGFBR1, and TGFBR2) should be obtained to establish whether aortic enlargement is occurring. 4,74 The final clinical management of patients with a specific gene mutation could be modified on the basis of these data, enabling personalized treatment that is independent of the native aortic diameters. 4,5,41,50 Only relatives in whom a causal mutation is excluded and the aortic size is within recommended diameters should reasonably undergo clinical and/or imaging screening every 2 to 5 years, until diagnosis is confirmed or ruled out. 5,76 The appropriate temporal interval for follow-up imaging, as well as the starting age for aortic surveillance, are also poorly defined. Generally, patients with NS-TAD are diagnosed on average 10 years older than patients affected by syndromic aortopathies, being also characterized by a lower annual aortic dilatation progression (0.5-0.7 mm/y). 59,60 It therefore seems reasonable to initiate the screening 15 to 10 years earlier than first aneurysm or when dissection or sudden death is recorded within the family. 60,79 A screening pathway based on these observations is proposed in Figure 3.
There are several additional factors that may influence our proposed screening algorithm. First, variable penetrance, which often characterizes NS-TAD forms, is a potential confounder. This results in intrafamilial variability, which is evident not only with reference to the aortopathy itself (severity, age of onset), but also with regard to other phenotypic manifestations. [65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80][81] The presence of associated features is certainly suggestive of having inherited the aortic condition along with the predisposition to the aortopathy, but the absence of these associated features does not eliminate the risk of having an underlying aortopathy. Second, women often demonstrate a lower lifetime risk of aortopathy,

Age (yrs) at dissecƟon
Ascending aorta diameter (mm) at dissecƟon Figure 2. Schematic representation of genetic mutations with age and ascending aorta diameter at dissection. The widening of the circles/lines represents SD in terms of age and diameters. Data are obtained from studies included in the systematic review. No numerical data were available for patients affected by aortic dissection regarding the genes NOTCH1 and MFAP5, and patients with MAT2A mutation did not experience aortic dissections. 1,36,43 developing the condition at a later age than men. 81 This phenomenon, known as sexual dimorphism, explains the apparent paradox of an affected teenager with an affected maternal grandfather but an unaffected mother with normal echocardiographic features. Third, the age at onset of the aortopathy may be important in the natural history of the disease. Ma et al 82 recently demonstrated that age at onset of aortic dissection is lower in families with a positive history for aortic dissection, therefore suggesting a prompt and more aggressive screening pathway in these families. A positive family history with an aortopathy occurring at younger ages confers a significantly increased risk of developing a new dissection in apparently unaffected family members. 81 The above findings are all important in guiding the proper screening and surveillance strategies.

Study Limitations
The most important limitation of the review is the uncertainty regarding the likely yield of new cases if a screening program were to be implemented. The studies identified in our searches were predominantly studies of familial aortopathy, and the prevalence of TAD in these populations will not reflect that for NS-TAD overall. Conversely, sporadic NS-TAD, which constitutes the majority (80%) of all cases also has a genetic component. 7   in sporadic NS-TAD. 83 This suggests that the relatives of patients affected by both familial and sporadic NS-TADs may benefit from screening. It also argues for use of nontargeted genetic screening tests such as exome or whole genome sequencing that will detect de novo or as-yet unrecognized common mutations. A second limitation is that there is currently no evidence to inform secondary prevention or intervention strategies in newly diagnosed NS-TAD, particularly where the aorta is phenotypically normal. Although the evidence presented supports the routine implementation of combined imaging and genetic testing in relatives of patients with NS-TAD, no study has proven that stratified treatment, independent of the native aortic diameter, will save lives. However, the stratified treatment of syndromic TAD is common practice, as this is known to prevent deaths from aortic disease. We suggest that the results of this review support the extension of similar programs to all patients with TAD. To address these limitations, we propose that further research should first establish the true prevalence of genetic abnormalities and phenotypic disease diagnosed by screening (genetic testing and imaging) all FDRs and SDRs of patients with both familial and sporadic NS-TAD. Further studies will be required to address uncertainty with respect to effectiveness, psychological impact, and the costs of lifelong screening in these groups. Finally, the heterogeneity of the included studies, the large period of publication across 3 decades, and the familial-based approach have limited our ability to analyze the impact of region or ethnicity in the risk of aortopathies and the related screening strategy.

Conclusions
The findings of this review support routine imaging and genetic testing of relatives of patients with nonsyndromic aortopathies. The evidence suggests that screening of FDRs and SDRs of patients affected by familial NS-TAD and FDRs of those affected by sporadic NS-TADs will result in significant numbers of patients with otherwise undiagnosed disease. Personalized screening programs determined by the subtype of NS-TAD and its related genetic mutation have the potential to benefit these patients. However, the diagnostic yield of available screening tests is unclear, as are the details of a screening program, and there is no existing evidence that routine screening and stratified treatment will have clinical or economic benefits. Further studies are required to address these knowledge gaps.
Mariscalco and Debiec had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Disclosures
Mariscalco declares support from Vascutek, an aortic prosthesis manufacturer, to attend scientific meetings. Murphy declares support from BHF chair of cardiac surgery, Vascutek for attendance at scientific meetings and financial support for educational activities. The remaining authors have no disclosures to report.

Rationale
Recent guidelines on diagnosis and management of thoracic aorta disease (TAD) have identified a knowledge gap with respect to the most effective screening modality for relatives of patients affected by non-syndromic TAD. Previous research has established a specific and clear screening pathway for syndromic TAD forms, including Marfan (MFS) and Loeys-Dietz (LDS) syndromes, and other similar connective tissue diseases.
Considering the incidence of NS-TAD and the impact of prompt diagnosis in improving clinical outcomes in TAD, we attempted to analyse the existing evidence that relates to screening modality and programs in relatives of patients affected by non-syndromic TAD.

Key points
Thoracic aortic disease is a term that essentially refers to an interrelated collection of pathologies that include thoracic aortic aneurysms (TAA) and aortic dissections (TADA). 1,2 TAA is often silent and commonly present as life-threatening emergencies, referred to as acute aortic syndromes. 1,2 In the United Kingdom (UK), over 6,500 deaths are attributable to TAD every year, and this number is increasing. 3,4 Attempts to formulate consensus statements and relevant guidelines have identified significant gaps in the knowledge with respect to the pathogenesis, appropriate management of, and configuration of clinical services for optimal treatment of aortic disease. This results in high variation in the diagnosis and management approach and regional differences in the quality of care and outcomes. 1,2,4 In particular methodology and modalities for genetic or imaging familial screening sof relatives of patients with non-syndromic forms of TAD (NS-TAD) are not well established. 1,2 To address this knowledge gap, we propose to undertake a systematic review of the existing literature that relates to genetic and/or imaging screening undertaken in relatives of patients with NS-TAD (diagnosed and/or operated on). Secondary aims are also to determine the effectiveness of screening in relatives of NS-TAD patients, and to catalogue existing evidence on genetic association in non-syndromic TAD.

Epidemiology and outcomes of TAD
The term "thoracic aortic disease" includes a wide range of aortic diseases with variable clinical presentations and prognosis. The Global Burden of Disease 2010 project demonstrated that the overall global death rate from aortic aneurysms and aortic dissection increased from 2.49 per 100000 to 2.78 per 100000 inhabitants between 1990 and 2010, with higher rates for men. 5 At the same time, admissions for thoracic aortic aneurysms have increased from 4.4 to 9.0 per 100000 in the UK, mainly due to an increase in proportion of elderly patients, over 75 years of age. 3 The epidemiology of TAD is difficult to establish since aortic diseases may be diagnosed after a long period of subclinical development or they may have an acute fatal presentation. In addition, the natural history of TAD remains poorly understood, and errors in the diagnostic process may account for deaths otherwise attributed to other diseases such as myocardial infarction or pulmonary embolism. TADs are usually asymptomatic until an acute complication occurs, requiring a prompt diagnosis and treatment in specialized centres. Management of TAD is complex and dictated by the size, extent and location of the disease condition as well as the underlying pathology (aneurysm or dissection). Options include conservative medical therapy (e.g. oral hypotensive agents such as beta blockers, ace-inhibitors, diuretics or statins) open surgical intervention, thoracic endovascular aortic repair (TEVAR), or hybrid procedures including epiaortic vessel debranching. 1,2 Early and late results also vary across centres and countries. In Europe and the wider world, mortality rates for operated type A acute aortic dissection range from 12% to 42%. 4,6-8 However, in some high-volume USA centres the mortality rate is lower, ranging from to 2 to 10%. 9,10 On the other hand, hospital mortality from elective nondissection surgery on the thoracic aorta ranges from 5% to 10%. 11 For patients suffering from an acute type B aortic dissection, mortality rates for medical treatment approach, endovascular and open surgical repair range from 3% to 20%. 12,13 Table 1 summarizes early and long-term mortality for treated TADs.

Forms of TAD
Currently TAD can be subdivided in two main entities: 1) Syndromic TAD 2) Non-syndromic TAD (NS-TAD) Up to 20% of individuals with TAD who do not present pathognomonic features of syndromic forms (especially MFS or LDS), have a family history of TAA and/or TADA. 22 Syndromic forms of TAD are associated with abnormalities of other organs, while those non-syndromic present manifestations limited to the thoracic aorta only. NS-TAD includes two distinct sub-groups: the familial (more than one family member is affected) and the sporadic TAD forms. 22 Table 2 summarizes syndromic and non-syndromic forms of TAD. 23

MFS (Marfan syndrome) FTAAD LDS (Loeys-Dietz syndrome) Vascular Ehlers-Danlos syndrome
Familial TAA Shprintzen-Goldberg syndrome Aneurysms-osteoarthritis syndrome BAV with thoracic aortic aneurysm Cutis laxa with aneurysm A genetic predisposition to the development of TAD in non-syndromic forms has been documented in 19% of patients, and patients with familial TAD are younger at the time of diagnosis that those with sporadic forms, but older when compared to syndromic TAD forms. 22 Previous studies have also suggested that 20% of NS-TAD patients referred for surgery have first-degree relatives similarly affected. 22,24 In majority of patients the familial NS-TAD is inherited as an autosomal-dominant disorder with decreased penetrance and variable expression. Several genes have been demonstrated to be involved NS-TAD (Table  3). 23,25,26

Imaging modality for screening TAD
Imaging techniques play a crucial role in the diagnosis, follow-up and management of TAD. Ultrasound, including transthoracic (TTE) and transoesophageal (TOE) echocardiograms, computed tomography (CT) and magnetic resonance (MR) can be used for the assessment of aneurysms and dissections located in the different segments of the thoracic aorta. All these imaging modalities have their strengths and limitations, and no single imaging modality has a perfect resolution (Table 4). 1,2,27 Page 6 _______________________________________________________________________________________________________________________________________________ The preferred imaging modality for screening of TAD has not yet been recommended in the international guidelines (ESC, AHA), and a variable combinations of imaging modalities at baseline and during follow-up have been reported. In addition, relationship between genetic and imaging screening modalities has not been elucidated in relatives of patients with NS-TAD.

Genetic screening for TAD
Establishing a specific genetic cause of NS-TAD is of paramount importance for defining the most appropriate management for the relatives of affected patients. Risk assessment and surveillance as well recommendations for specific medical and surgical management are based on the gene identification. Specific genes have been identified, each of them are involved in specific aortopathy pathways (Table 3). Multi-gene panel, single-gene testing and genomic sequencing all can be utilized as evaluation strategy to identify the genetic cause of NS-TAD formm. 26 For some genes, specific recommendations exist in order to tailor the most appropriate clinical and/or surgical intervention. In patients with ACTA 2 gene mutations, elective surgical repair is advisable when the diameter of the ascending aorta/aortic root reaches 4.5 cm; 28

The knowledge gap
The 2014 European Society of Cardiology (ESC) Guidelines for the management of NS-TAD include a level I recommendation for the screening of first-degree relatives of patients with TAA and /or TADA to identify those with asymptomatic disease, and for referring the patient to a geneticist for family investigation, once a familial NS-TAD from is recognized. 1 However, the evidence to support these recommendation is level C, based on the

Why it is important to do this review
Compared to syndromic TAD forms (i.e. Marfan or Loeys-Dietz syndromes) which are characterized by relevant physical features, therefore alerting clinicians to the underlying aortopathy, non-syndromic (NS) TAD forms lack of clear external physical signs, and are characterised by silent aneurysm formation and dissection. 22,24 Thoracic aortic disease (TAD) have high mortality, and early recognition is essential in order to establish a prompt clinical and surgical management, 1,2 therefore identifying as early as possible those who would benefit from prompt treatment and preventive measures.

OBJECTIVES
The overarching aim of the present review is to determine the effectiveness of screening of asymptomatic relatives of NS-TAD probands, highlighting the incidence and prevalence of TAD in this population. Secondary objectives will be to catalogue all screening modalities (both genetic and imaging) adopted in the above relatives, and to assess the effectiveness or cost-effectiveness of screening.

Hypothesis
It is our hypothesis that systematic screening of first-and second-degree relatives of patients affected by NS-TAD will provide a substantial benefit in identifying silent TAD and preventing related death.
Furthermore systematic review of the existing evidence may help with clarifying the best cost-effective screening modality or combination of modalities (genetic vs imaging) and/or imaging tools (TTE vs CT vs MRI), and may contribute to create a catalogue with all the known genetic markers associated with TAD.

Aims
The aims of the present review will be: 1. To summarise published studies that have considered the screening in relatives of patients with by NS-TAD; 2. To estimate the incidence and prevalence of TAD in family members of patients with NS-TAD of silent and undiagnosed disease of thoracic aorta (TAA and TADA); 3. To provide a defined screening strategy to identify potential individuals affected by TAD who will benefit the most from tailored clinical or surgical managements; 4. To provide a comprehensive list of genes, which can be utilized as risk assessment in family members of a proband with NS-TAD.

Types of studies
We will consider clinical studies that have performed genetic and/or imaging evaluation of relatives of patients affected by NS-TAD. The following types of studies will be analysed: 1. Clinical randomised trials; 2. Controlled before-and-after studies; 3. Prospective and retrospective cohort studies; 4. Cross-sectional studies; 5. Case-control studies; 6. Case series. Study design features will be assessed according to established criteria from the Cochrane Handbook. 30 In addition, inclusion and exclusion criteria for qualitative and quantitative analyses will be presented according to PICOS criteria.

Study exclusion criteria
Exclusion criteria will include: 1. Studies where screening is based on clinical patient evaluation only; 2. Studies where screening does not include genetic patient evaluation and/or patients are not subjected to recognised imaging modality such as TTE/TOE, CT and MRI of the thoracic aorta; 3. Studies where screening is not based on prospective recruitment/analysis of the proband relatives; 4. Studies where screening involved patients without clear differentiation from syndromic forms; 5. Repeat publications of the same analysis or dataset; 6. Conference abstracts; 7. Editorials & opinion pieces; 8. Books or grey literature.

Types of participants
Relatives of probands with a diagnosis of NS-TAD, including aneurysm, aortic rupture, acute/chronic aortic dissection, intramural hematoma, and penetrating ulcer of the thoracic aorta.

Variable definitions
 Familial non-syndromic TAD will be defined as those occurring in patients having 1 or more first-generation relatives with an aortic aneurysm and no history of MFS or any other connective tissue disease (Table 2). 22  Sporadic TAD will be defined as those occurring in patients apparently without another relative with TAD.  Patients affected by TAD will be considered in the entire family pedigree, and will be defined as those individuals having a diagnosis of TAD. Their percentage will be considered in the obtained family pedigree.
 Diagnosis of TAD (phenotype) will be considered if confirmed by imaging (TTE and/or CT and/or MRI), postmortem examination or intraoperative findings. Sudden deaths will be excluded from TAD diagnosis.  Percentage (%) of observed TAD will be calculated from the total number of relatives in the entire pedigree.  Patients defined as eligible for screening (genetic and/or imaging) will include first-and second-degree relatives of a proband with NS-TAD; spouse and deceased patients will be included if blood/tissue samples were available for analysis.  Patients screened will be defined as those having had prospective genetic screening and imaging studies (TTE and/or CT and/or MRI). Patient deceased will be included in the "patient screened category" if they had blood or tissue collected at the time of operation , which allowed for subsequent genetic analysis.  Percentage (%) of screened patients will be calculated from the number of patients considered eligible for screening.  Proband (index patient) will be defined as the first family member affected by NS-TAD. It will be denoted as shaded square (male) or circle (female) in the family pedigree marked by an arrow.
 Penetrance (%) will be defined as: n. of patients affected by TAD positive for the gene mutation Subjects with positive gene mutation  First-degree relatives (FDR) of the proband will include: 1) Parents (father and mother) 2) Child (daughter and son) 3) Siblings (brother and sister).
 Thoracic aortic dissection (TADA) category will include type A and B acute or chronic forms as well as other acute aortic syndromes (rupture, intramural hematoma, penetrating ulcer).

Exposures of Interest
The primary exposure of interest will be a disease of the thoracic aorta (aneurysm and dissection).

Types of outcome measures
• The primary outcome will be new diagnosis of TAD, including aneurysms or dissections, in relatives of patients with NS-TAD forms.
• Secondary outcome will include: a. Gender TAD preponderance; b. Rate between TAA and TADA in the NS-TAD form; c. Age at diagnosis of TADA; d. Concomitant vascular/cardiac associated diseases; e. Concomitant associated clinical features; f. Genetic risk assessment with the penetrance of the NS-TAD form; g. Cost-effectiveness of adopted imaging modality.

Search strategy
We will search the following databases (from inception to 31 December 2017): No language restriction will be applied. We also anticipate that articles not in English will be translated using Google Translate® which is a free, Web-based program with a reputation for accurate, natural translation. 31,32

Searching other resources
A systematic search in the Online Mendelian Inheritance in Man (OMIM) database (http://www.omim.org/) will be also performed through December 2017, using similar terms of the below literature search.
Finally, we will check references of all identified studies, relevant review articles, and current treatment guidelines for further literature. These searches will be limited to the 'first generation' reference lists.

Results of the scoping search
A preliminary scoping search (PUBMED) using the terms (aorta, thoracic) or (aortic aneurysm) or (aortic dissection) AND (relatives) or (pedigree) or (siblings) and (screening) and (humans) accounted for 1,022 sources.

Selection of studies (screening-eligibility-inclusion)
Two authors (G.M. and D.R.) will screen all titles and abstracts of papers identified for relevance to the review aims (electronic search). An independent search with the review of all articles will be conducted by a third review (G.J.M.). Studies clearly not meeting the eligibility criteria will be excluded at this stage. Remaining studies will be assessed on the basis of their full text for inclusion or exclusion using the criteria indicated above. At this stage, two reviewers (G.M. and D.R.) will independently assess eligibility. Disagreements will be resolved by consensus in discussion with a third reviewer (G.J.M.). Numbers of studies assessed, included and excluded will be recorded. Duplicate reporting of studies will be carefully assessed and indicated.

Qualitative analysis
Two investigators independently will appraise all articles that will met inclusion criteria, and study quality will be assessed using the Newcastle-Ottawa Scale, and the U.S. Preventive Services Task Force (USPSTF). 33,34 Methdodological quality will be also assessed considering the Cochrane Risk of Bias toll. 35 Disagreement about critical appraisal will be resolved by discussion. The qualitative analysis will help to explore questions such as how patient selection, treatment and type of study may have influenced the primary effect estimate. In addition, the following questions will be considered for a qualitative analysis: 1. Was the study population well described? 2. Were the outcomes of interest clearly defined? 3. Were the exposures of interest (primary and secondary) well defined? 4. Does the article state both inclusion and exclusion criteria? 5. Were the analysed variables clearly defined? 6. Was the screening prospectively conducted? 7. Were relatives prospectively invited and subject to screening (genetic and/or imaging)?

Data extraction and management
Two authors (G.M. and D.R.) will extract selected data from eligible studies, which will be subsequently checked by a third author (G.J.M.). The following data will be collected and tabulated with Microsoft Excel ( Rate of registered sudden death; age (years) at diagnosis for patients with TADA (mean and range); gender preponderance; rate and type of concomitant associated cardiac or vascular diseases; rate and type of concomitant associated clinical features; penetrance; identification of genetic mutation. 5. Screening modality: Type of adopted genetic screening; type of imaging modality adopted for screened. Two authors (G.M. and R.D.) will perform data extraction independently. Data will be extracted onto study specific data extraction form. Disagreements will be resolved by consensus between the authors or by discussion with a third author where necessary (G.J.M.). A second check of all data entry will be performed in order to avoid discrepancies. Missing data will be requested from study authors. If data are unclear, missing, or presented in a form that is unable to be reliably extracted, authors will be contacted to assist in the process.
The corresponding author will be initially contacted by email, with the first author (if not the corresponding author) copied into all correspondence. If email addresses are not available, authors will be contacted by phone. Authors will be given seven days to respond to emails, after which they will be followed up with a phone call and an additional email. If no responses are received after an additional seven days, another phone call will be made to contact the author. Other attempt will occur for other seven days; thereafter the authors will be classified as uncontactable.

Measures and data representation
A narrative synthesis of the included studies will be provided, focusing on the effectiveness of genetic and/or imaging in the new diagnosis of TAD, including aneurysms or dissections, in relatives of patients with NS-TAD forms. Detailed tables of the findings from the included studies will be provided, with reference to the type of study (i.e. randomized, cohort studies, case control studies...), origin (country), the study period (year), the inclusion/exclusion criteria, type of analysed outcomes, and modality of screening adopted. In addition, additional tables will be provided listing relevant characteristics of each study.

Data analysis
All extracted data will be tabulated with Microsoft with Microsoft Excel (Microsoft Corporation, Redmond, WA). Percentage for screened, eligible patients as well subjects affected by TAA and/or TADA will be provided. Percentages of other associated concomitant vascular and cardiac disease will be listed as well concomitant associated clinical features.

COMPETING INTERESTS
The authors declare that they have no competing interests.

METHODS
Protocol and registration 5 Indicate if a review protocol exists, if and where it can be accessed (e.g., Web address), and, if available, provide registration information including registration number.

(Data S1)
Eligibility criteria 6 Specify study characteristics (e.g., PICOS, length of follow-up) and report characteristics (e.g., years considered, language, publication status) used as criteria for eligibility, giving rationale.

(Data S1)
Information sources 7 Describe all information sources (e.g., databases with dates of coverage, contact with study authors to identify additional studies) in the search and date last searched.

Search
8 Present full electronic search strategy for at least one database, including any limits used, such that it could be repeated. 4 Study selection 9 State the process for selecting studies (i.e., screening, eligibility, included in systematic review, and, if applicable, included in the meta-analysis).

4,5 (Data S1)
Data collection process 10 Describe method of data extraction from reports (e.g., piloted forms, independently, in duplicate) and any processes for obtaining and confirming data from investigators.

4,5
(Data S1) Data items 11 List and define all variables for which data were sought (e.g., PICOS, funding sources) and any assumptions and simplifications made.
4,5 (Table S2) Risk of bias in individual studies 12 Describe methods used for assessing risk of bias of individual studies (including specification of whether this was done at the study or outcome level), and how this information is to be used in any data synthesis. 6 (Table S9) Summary measures 13 State the principal summary measures (e.g., risk ratio, difference in means). 5 Synthesis of results 14 Describe the methods of handling data and combining results of studies, if done, including measures of consistency (e.g., I 2 ) for each meta-analysis. 6 Risk of bias across studies 15 Specify any assessment of risk of bias that may affect the cumulative evidence (e.g., publication bias, selective reporting within studies). 6 Additional analyses 16 Describe methods of additional analyses (e.g., sensitivity or subgroup analyses, meta-regression), if done, indicating which were pre-specified. 6

Study selection
17 Give numbers of studies screened, assessed for eligibility, and included in the review, with reasons for exclusions at each stage, ideally with a flow diagram. 7 Study characteristics 18 For each study, present characteristics for which data were extracted and provide the citations. 7 Risk of bias within studies 19 Present data on risk of bias of each study and, if available, any outcome level assessment (see item 12). 7 Results of individual studies 20 For all outcomes considered (benefits or harms), present, for each study: (a) simple summary data for each intervention group (b) effect estimates and confidence intervals, ideally with a forest plot.

Intervention
Screening using the genetic and/or imaging modalities, including transthoracic echocardiography, computed tomography, and magnetic resonance -

Comparator
The screening interventions listed above versus each other or versus no intervention -

Outcomes
Primary: new diagnosis of TAD (aortic aneurysm and dissection) in first-, second-, and third-degree relatives Secondary: effectives of screening modality (eligible vs screened relatives), disease-specific mortality, disease specific genetic mutation, cost-effectiveness, age and aortic diamters at dissection/grow rate -

Class Level of evidence Familial thoracic aortic aneurysm and dissections
Aortic imaging is recommended for first-degree relatives of patients with thoracic aortic aneurysm and/or dissection to identify those with asymptomatic disease.

I B
If the mutant gene (FBN1, TGFBR1, TGFBR2, COL3A1, ACTA2, MYH11) associated with aortic aneurysm and/or dissection is identified in a patient, first-degree relatives should undergo counseling and testing. Then, only the relatives with the genetic mutation should undergo aortic imaging.

I C
If one or more first-degree relatives of a patient with known thoracic aortic aneurysm and/or dissection are found to have thoracic aortic dilatation, aneurysm, or dissection, then imaging of second-degree relatives is reasonable

IIa B
Sequencing of the ACTA2 gene is reasonable in patients with a family history of thoracic aortic aneurysms and/or dissections to determine if ACTA2 mutations are responsible for the inherited predisposition

IIa B
Sequencing of other genes known to cause familial thoracic aortic aneurysms and/or dissection (TGFBR1, TGFBR2, MYH11) may be considered in patients with a family history and clinical features associated with mutations in these genes

IIb B
If one or more first-degree relatives of a patient with known thoracic aortic aneurysm and/or dissection are found to have thoracic aortic dilatation, aneurysm, or dissection, then referral to a geneticist may be considered

Bicuspid aortic valve and thoracic aortic disease
First-degree relatives of patients with a bicuspid aortic valve, premature onset of thoracic aortic disease with minimal risk factors, and/or a familial form of thoracic aortic aneurysm and dissection should be evaluated for the presence of a bicuspid aortic valve and asymptomatic thoracic aortic disease I B

Familial thoracic aortic aneurysm and dissections
It is recommended to investigate first-degree relatives (siblings and parents) of a subject with TAAD to identify a familial form in which relatives all have a 50% chance of carrying the family mutation/disease

I C
Once a familial form of TAAD is highly suspected, it is recommended to refer the patient to a geneticist for family investigation and molecular testing I C Variability of age of onset warrants screening every 5 years of 'healthy' at-risk relatives until diagnosis (clinical or molecular) is established or ruled out I C In familial non-syndromic TAAD, screening for aneurysm should be considered, not only in the thoracic aorta, but also throughout the arterial tree (including cerebral arteries) IIa C

Bicuspid aortic valve and thoracic aortic disease
Because of familial occurrence, screening of first-degree relatives should be considered IIb C Figure S1. PRISMA flow diagram of search strategy (through December 31, 2017) Articles reviewed for more detailed evaluation (including cross references)

2642
Full-text articles assessed for eligibility 164 53 Studies included for qualitative synthesis Records excluded after duplicates removed, title review (case reports, editorials, syndromic forms of TAD, no relative screening)

10255
Titles excluded (case reports, case series, editorials, reviews, no relative screening, syndromic forms of TAD)