A De Novo Missense MYLK Variant Leading to Nonsyndromic Thoracic Aortic Aneurysm and Dissection Identified by Segregation Analysis

Nonsyndromic hereditary thoracic aortic aneurysm and dissection (TAAD) is an autosomal dominant disease; however, it is frequently difficult to identify the causative genes. We report in this study a 33-year-old Japanese male with TAAD (Stanford type A) that is complicated with severe aortic regurgitation. There was no family history of aortic diseases in the patient nor any specific clinical features suggestive of connective tissue diseases, such as Marfan syndrome. Genetic testing identified candidate causative variants in two different genes: MYLK (c.4819G > A, p.[Gly1607Ser]) and FBN1 (c.365G > A, p.[Arg122His]). Familial cosegregation analysis revealed that the novel de novo MYLK variant was present only in the proband, and the FBN1 variant was also found in his nonaffected mother, and thus the MYLK variant was classified as likely pathogenic. MYLK is a causative gene for nonsyndromic TAAD that requires careful management; however, the number of reports is limited. Accumulating data on the pathogenicity of rare variants by performing a comprehensive pedigree analysis would help establish better treatment strategies for life-threatening hereditary TAAD cases.


Introduction
Nonsyndromic hereditary thoracic aortic aneurysm and dissection (TAAD) is an autosomal dominant disorder with a wide range of clinical variabilities and incomplete penetrance [1]; however, its natural history of progress is still unknown.Most nonsyndromic TAAD cases are diagnosed due to early-onset age or family history, and the identifcation of pathogenic variants may provide prognostic and risk stratifcation information to the proband and their family members.Prophylactic aortic surgery is advised for the most severe type, which can be life-threatening, based on the specifc genetic variant and aortic diameter [2][3][4].
Recently, the discovery of multiple genes associated with nonsyndromic TAAD was accelerated by next-generation sequencing technology [5][6][7], and this trend has made genetic testing indispensable for diagnosing genetic diseases and in their subsequent therapeutic management.Currently, rare variants and polymorphisms in FBN1 (OMIM * 134797), which cause Marfan syndrome (MFS), have also been reported to be involved in nonsyndromic TAAD [8,9]; however, when assessing the pathogenicity of unclassifed variants, it is important to ensure that preventive measures are taken.

Segregation Analysis.
Segregation analysis of the candidate variant using Sanger sequencing was carried out in additional family members.For genetic testing for MYLK and FBN1 variants, genomic DNA isolated from the white blood cells of the proband and his parents was used.In order to amplify exon 28 of MYLK and exon 5 of FBN1, intron-specifc primers fanking exons were designed for polymerase chain reaction (PCR).PCR was carried out using the following primers located in exon 28 of MYLK (forward, 5′-GGGCTTTCCTGCCAATTTCTCCTACCCCC-3′, and reverse, 5′-GGGGCAGGATTCAAACCTGGG-AAGTCTTC-3′) and exon 5 of FBN1 (forward, 5′-GG-AGTCAAGCAAGATGGAGC-3′, and reverse, 5′-AGCTGACACTACTTTTCCATTCT-3′).All sequencing procedures were performed using forward and reverse primers, as mentioned previously [14].

Case Presentation.
Te patient was a 33-year-old Japanese male without apparent medical history.He suddenly developed chest pain and was transferred to a nearby hospital in May 2021.Computed tomography (CT) showed a 45 mm dilatation of the ascending aorta, but aortic dissection was not widely suspected.He kept experiencing symptoms of shortness of breath and chest pain on exertion afterward, and 3 months later, he returned to the outpatient clinic.Cardiac enlargement was observed on chest X-ray, and severe aortic insufciency with ascending aorta enlargement was shown on echocardiogram.Contrastenhanced CT revealed a Stanford type A false lumen aortic dissection that extended from the aortic root to the aortic arch without organ malperfusion.Te maximum diameter of the ascending aorta increased to 55 mm compared to 3 months ago (Figure 1(a)).He was transferred to our university hospital, where he was immediately admitted for further evaluation, and transesophageal echocardiogram showed a false lumen at the aortic root and severe aortic insufciency due to the enlargement of aortic valve ring (Figure 1(b)).Symptoms of heart failure were seen, and the diameter of the ascending aorta increased over the past few months; therefore, elective surgery was performed.Valvesparing aortic root replacement (David procedure) and aortic valvuloplasty were performed.Te thickness of the ascending aortic wall was increased with elastic fber degeneration and collagen deposition, and the number of vasa vasorum in the adventitia was increased compared to that in the control specimen from a heart transplant recipient (Figures 1(c) and 1(d)).Moreover, compared with the control, the phosphorylation levels of Smad2 in the adventitia and ERK1/2 in the media were signifcantly upregulated (Figures 1(e) and 1(f )), which suggests the activation of the transforming growth factor-β (TGF-β) signaling pathways in the patient.

Segregation Analysis.
Tere was no apparent family history of connective tissue diseases (CTDs) or cardiovascular diseases observed in the patient (Figure 2 .We conducted the familial cosegregation analysis using Sanger sequencing to investigate the impact of these variants on arteriopathy in this family.Consequently, the missense variant of MYLK was detected only in the proband and not in their parents, and therefore this was considered to be a de novo variant (Figure 2(b)).Te patient and his mother carried the missense variant of FBN1, whereas the patient's father had neither (Figure 2(c)).Te patient's mother was 62 years old, and no enlargement of the sinus of Valsalva was seen on echocardiography (31 mm; aortic root Z-score [15], 0.68).
According to the American College of Medical Genetics and Genomics (ACMG) guidelines [16] and the Clinical Genome Resource (ClinGen) recommendations [17], the MYLK and FBN1 variants were classifed as likely pathogenic (PM1, PM2_Supporting, PM6_Supporting, PP3, and PP4) and VUS, respectively, in the present case.

Discussion
A young patient with nonsyndromic TAAD was reported in this study.Gene panel sequencing for TAAD-associated genes identifed two candidate causal missense variants in MYLK and FBN1.FBN1 is the causative gene for MFS, the most common inherited form of syndromic TAAD, but FBN1 rare variants and polymorphisms have been also reported to contribute to nonsyndromic TAAD [8,9].Conversely, pathogenic variants of MYLK cause nonsyndromic TAAD, and the natural history and appropriate treatment strategy remain elusive [10].
Personalized aortic surveillance and clinical management would be supported by validation of pathogenicity of gene variants.In the present case, c.4819G > A in MYLK was a novel de novo heterozygous variant, and based on the ACMG guidelines and the ClinGen recommendations, it is classifed as likely pathogenic.Although most MYLK pathogenic variants are truncating variants (e.g., frameshift and nonsense variants) which resulted in premature truncation and nonsense-mediated decay and are thought to cause disease as a consequence of haploinsufciency, only a few pathogenic missense variants of MYLK have been previously reported, two of which remarkably reduce MLCK kinase activity in vitro [10,11]: c.5275T > C (p.Ser1759Pro) in the calmodulin binding site and c.4471G > T (p.Ala1491Ser) in the kinase domain (ClinVar database accession numbers are RCV000023044.4and RCV000855690.3,respectively).Although c.4819G > A located in the MLCK kinase domain may also have a loss-offunction efect in vitro, further research is required in order to fully understand the variant functional efect, since missense variants were associated with a higher risk of frst aortic event (elective aortic aneurysm surgery or acute aortic dissection) than truncating variants [18].
Te in vivo mechanisms through which MYLK pathogenic variants cause aortopathy are completely unknown.Activation of ERK1/2 and Smad2 and enhanced vasa vasorum formation were observed in proband's aortic wall; however, these changes might be infuenced by aortic dissection, while the increased vascularity in the aortic media has been reported from patients who had nondissecting thoracic aneurysm and MYLK pathogenic variants [10,19].It is interesting to note that MYLK pathogenic variants can cause aortic dissection with little to no enlargement of the aorta [10].Te ascending aorta was mildly dilated as seen on CT at the frst examination (diameter 45 mm) in the present case, and 3 months later, it expanded to 55 mm.Although the exact onset time of aortic dissection was uncertain, his aortic size before dissection might have been much smaller than at dissection, since aortic dilatation occurs rapidly in aortic dissection [20].
c.365G > A in FBN1 might serve as a modifer for the disease.Te penetrance of FBN1 pathogenic variants is reported to be 100% [21,22], and c.365G > A has been observed in patient with nonsyndromic TAAD [23]; however, currently, there are not enough data to determine the impact of c.365G > A variant on FBN1 protein function and TAAD pathogenesis, which includes MFS.In the present family, the proband did not have pear-shaped aortic root enlargement typical for MFS, and his mother who has c.365G > A did not show any characteristic features of MFS which includes aortic aneurysm.Tese results lead us to the conclusion that c.4819G > A in MYLK, as opposed to c.365G > A in FBN1, had a stronger efect on the development of aortic dissection in the proband.
Because there are few reports on MYLK pathogenic variants, the best course of treatment and indications for prophylactic aortic surgery have not, to date, been convincingly established.Although genetic testing has grown more accessible, faster, and afordable, uncertainty in variant interpretation can result in incorrect diagnosis and prevent appropriate therapy and genetic counseling, especially when multiple candidate variants are identifed as it happened this time.Better treatment options for rare diseases like nonsyndromic hereditary TAAD could be established by accumulating data on the pathogenicity of rare variants by performing a thorough pedigree analysis.
In conclusion, we reported a Japanese case of nonsyndromic TAAD with a novel likely pathogenic variant of MYLK (c.4819G > A), simultaneously carrying FBN1 c.365G > A VUS.When genetic testing identifes multiple candidate disease-causal variants, segregation analysis may help detect high-risk patients in which careful treatment strategies are needed.

Figure 1 :
Figure 1: Japanese case of familial TAAD presenting with aortic disease.(a) Dissection from the aortic root to the aortic arch as shown on contrast-enhanced computed tomography (sagittal cross section).Te maximum diameter of the ascending aorta was 55 mm and the sinus of Valsalva was 36 mm.(b) Transesophageal echocardiogram reveals a fap in the aortic root and severe aortic insufciency.(c, d) Histological analysis with Elastica van Gieson stain and Masson's trichrome stain in the ascending aorta of the patient (MT65) and a control undergoing heart transplant (MT21).Abundant vasa vasorum in the patient's adventitia was observed (blue arrowheads).(e, f ) Immunostaining for phosphorylated ERK1/2 (p-ERK1/2) and phosphorylated Smad2 (p-Smad2) in the ascending aorta of the patient (MT65) and a control undergoing heart transplant (MT21).TAAD, thoracic aortic aneurysm and dissection; LA, left atrium; AV, aortic valve; LV, left ventricle; I, intima; M, media; A, adventitia.

Figure 2 :
Figure 2: Segregation analysis in this case.(a) Pedigree in this proband (III-1).A black symbol indicates that an individual is afected with thoracic aortic aneurysm and dissection, and "d" indicates age at death.Square, male; circle, female; P and arrow, proband; slash line, died; AD, aortic dissection.(b, c) A heterozygous single-base substitution (c.4819G > A, black arrow) in the MYLK gene and (c.365G > A, black arrowheads) in the FBN1 gene have been identifed by sequencing analysis, which shows that the proband alone has the MYLK variant (black arrow), whereas the proband and his unafected mother both carry the FBN1 variant (black arrowheads).
(a)), as well as no physical examination fndings suggestive of CTDs, such