The Association of Novel Single-Nucleotide Variants in the Collagen Matrix-Encoding Gene PRDM5 with Aortic Aneurysmal Disease

Thoracic aortic aneurysms are clinical conditions that are associated with severe clinical endpoints including dissection and rupture, potentially leading to sudden death. Contrary to their abdominal counterparts, thoracic aortic aneurysms are well-recognized to have a genetic basis underlying their development. Among all patients with aneurysmal disease who underwent clinical genetic screening in our program (N = 145), two patients were found to have variants of uncertain significance (VUS) in the PRDM5 gene. This gene is responsible for multiple regulatory functions in extracellular matrix development, and this is the first report, to our knowledge, to associate this gene with aortopathy.


Introduction
Thoracic aortic aneurysms (TAAs) are uncommon clinical findings in cardiovascular medicine that may be defined as a dilation of all three layers of the aortic arterial wall with at least a 50% increase in diameter when compared to the contiguous aorta.The current estimated prevalence is 6 persons per 100,000 each year.Symptoms of TAA vary, with many being clinically silent prior to their eventual discovery on routine imaging [1].Aneurysm formation is gradual and painless, which results in 95% of all cases remaining undetected until an acute aortic event such as dissection or rupture occurs, or until the expansion results in the compression of nearby structures [2].The current understanding of TAA pathogenesis is limited, and the search for the etiology behind TAA development is heavily dependent on genetic testing.Genetic influence is evident in TAA expression, with as many as 20% of affected individuals having a first-degree relative with a dilated thoracic aorta [3].The life-threatening complications of TAAs justify the importance of identifying genetic influences potentially responsible for aneurysmal development.
Suspected familial TAAs can be described as syndromic (20% of cases) or non-syndromic (80%).Syndromic classification includes aneurysm secondary to Marfan syndrome (MFS), Ehlers-Danlos syndrome (EDS), or Loeys-Dietz syndrome (LDS), while non-syndromic is further subdivided into sporadic and familial causes [2].Syndromic causes of TAA result in cystic medial degeneration, a process that involves the degeneration of the elastic medial layer of the aorta.This process results in a loss of wall integrity and subsequent aneurysm formation [4].Currently, 11 genes have sufficient clinical and scientific data to establish a highly penetrant risk for TAA and acute aortic dissections in patients with or without syndromic features.These 11 genes encode proteins involved in vascular smooth muscle cell contraction, extracellular matrix adhesion, transforming growth factor β signaling, or smooth muscle cell metabolism.These include the FBN1, LOX, MYH11, ACTA2, MYLK, PRKG1, COL3A1, TGFBR2, TGFBR1, TGF-β2, and SMAD3 genes.Disease in families with an underlying genetic mutation in the absence of a genetic syndrome is typically inherited in an autosomal dominant fashion with decreased penetrance [5].

Case 1
A 60-year-old Caucasian female with a history of a bicuspid aortic valve and ascending thoracic aortic aneurysm presented to our practice for a cardiac evaluation.Transthoracic echocardiogram (TTE) from an outside institution two years prior revealed an ascending aorta measuring 45 mm.At the time of our initial evaluation our patient was clinically asymptomatic and lived an active lifestyle without limitations.Initial physical examination was significant for a 2/6 graded holosystolic murmur over the right upper sternal border with radiation to the carotid arteries.To assess aortic valve function and the aortic anatomy, a TTE and computed tomography angiography (CTA) were performed.TTE revealed a bicuspid aortic valve with moderate calcific restriction of the superior leaflet as well as mild aortic valve insufficiency.Computed tomography angiography (CTA) revealed an ascending aorta measuring 46 × 45 mm with no other anomalies (Figure 1).We advised clinical genetic testing to exclude high-risk pathogenic mutations and to guide optimal clinical management.
Life 2023, 13, x FOR PEER REVIEW 2 of vascular smooth muscle cell contraction, extracellular matrix adhesion, transforming growth factor β signaling, or smooth muscle cell metabolism.These include the FBN1 LOX, MYH11, ACTA2, MYLK, PRKG1, COL3A1, TGFBR2, TGFBR1, TGF-β2, and SMAD3 genes.Disease in families with an underlying genetic mutation in the absence of a genetic syndrome is typically inherited in an autosomal dominant fashion with decreased pene trance [5].

Case 1
A 60-year-old Caucasian female with a history of a bicuspid aortic valve and ascending thoracic aortic aneurysm presented to our practice for a cardiac evaluation.Transthoracic echocardiogram (TTE) from an outside institution two years prior revealed an ascending aorta measuring 45 mm.At the time of our initial evaluation our patient was clinically asymptomatic and lived an active lifestyle without limitations.Initial physical examination was significan for a 2/6 graded holosystolic murmur over the right upper sternal border with radiation to the carotid arteries.To assess aortic valve function and the aortic anatomy, a TTE and computed tomography angiography (CTA) were performed.TTE revealed a bicuspid aortic valve with moderate calcific restriction of the superior leaflet as well as mild aortic valve insufficiency Computed tomography angiography (CTA) revealed an ascending aorta measuring 46 × 45 mm with no other anomalies (Figure 1).We advised clinical genetic testing to exclude high risk pathogenic mutations and to guide optimal clinical management.

Case 2
A 62-year-old Caucasian female with no significant medical history was referred to our practice for evaluation following recent cardiovascular events in her family.A review of prior records was notable for an ascending aorta measuring 39 mm in 2020 on a EBCT and subsequently 40 mm on a CT scan performed in 2021 for a pulmonary evaluation.A transthoracic echo in our program revealed no anomalies of the aortic valve, with a mildly enlarged ascending aorta.We proceeded with a thoracic CTA, which measured her as cending aortic aneurysm at 41.5 mm, notable for a 2 mm increase in size of the aorta ove three years (Figure 2).

Case 2
A 62-year-old Caucasian female with no significant medical history was referred to our practice for evaluation following recent cardiovascular events in her family.A review of prior records was notable for an ascending aorta measuring 39 mm in 2020 on a EBCT and subsequently 40 mm on a CT scan performed in 2021 for a pulmonary evaluation.A transthoracic echo in our program revealed no anomalies of the aortic valve, with a mildly enlarged ascending aorta.We proceeded with a thoracic CTA, which measured her ascending aortic aneurysm at 41.5 mm, notable for a 2 mm increase in size of the aorta over three years (Figure 2).The presenting TAA sizes of 4.6 cm and 4.15 cm with a mean of 4.35 cm +/− 0.25 cm found in our PRDM5 patients were comparable to the size seen in patients with Marfan TAAs secondary to known pathogenic mutations in FBN1.In a sample of 78 patients with Marfan syndrome, notably due to mutations in the FBN1 gene, the average aneurysm diameter at presentation was 4.54 ± 1.3 cm, creating a presenting range of 2.2-8.3 cm [6].

Materials and Methods
Patients in our program with a history of aneurysmal or dissection diseases underwent clinical genetic testing (n = 145) to assess the mutation status of genes associated with vasculature, extracellular matrix, and aneurysmal/dissection disease to guide clinical management.Those with mutations in PRDM5 (n = 2) are included as subjects in this study.Patients in this report underwent a clinical evaluation of thoracic aortic aneurysms and cardiac structure function using standard transthoracic echocardiography and thoracic CT angiographic techniques.

Materials and Methods
Patients in our program with a history of aneurysmal or dissection diseases underwent clinical genetic testing (n = 145) to assess the mutation status of genes associated with vasculature, extracellular matrix, and aneurysmal/dissection disease to guide clinical management.Those with mutations in PRDM5 (n = 2) are included as subjects in this study.Patients in this report underwent a clinical evaluation of thoracic aortic aneurysms and cardiac structure function using standard transthoracic echocardiography and thoracic CT angiographic techniques.

Case 1
We identified a variant of unknown significance (VUS), p.R83H variant (also known as c.248G>A), located on coding exon 3 of the PR/SET domain 5 (PRDM5) protein coding gene.A search of the gnomAD database did not reveal this VUS, reducing the likelihood that this is a cold variant or common finding [14].This single nucleotide variant (SNV) resulted in an arginine-to-histidine replacement at codon 83, two amino acids with similar properties (Table 1).The Grantham Score, which helps predict the effect of substitutions between amino acids, was measured at 29.This low score suggests less evolutionary distance.This mutation provides potential for the disruption of extracellular matrix components through the disruption of the secondary structure, hydrophilicity, and polarity of PRDM5.We identified a p.E129A variant (also known as c.386A>C) located on coding exon 4 of the PRDM5 gene.A search of the gnomAD database did not reveal this VUS, reducing the likelihood that this is a cold variant or common finding [14].This SNV resulted in a change from glutamic acid to alanine at codon 129, two amino acids with differing properties (Table 2).The Grantham Score was moderately radical, at 107.Though this variant was predicted to be tolerated by in silico analysis, this variation provided potential for the structural disruption of extracellular matrix components through changes in the PRDM5 secondary structure, hydrophilicity, and polarity.
Following the sequencing of all 35 genes in the TAAD panel, PRDM5 was the only gene demonstrating a genetic variation in these cases, making this report the first to associate the clinical phenotype of aortopathy and single nucleotide variants in PRDM5.We cannot attribute causation, as this requires further detailed characterizations of these SNV regarding protein structure, resultant biochemistry, and demonstration of the tissue level effect.However, we anticipate that our work will generate interest in these mechanistic studies to demonstrate causal effects between genetic variants in PRDM5 on tissue changes within the extra cellular matrix that subsequently lead to aortopathy.

Discussion: PRDM5
The identification of genetic variants that are responsible for the hereditary development of TAAs is necessary for establishing molecular mechanisms that are responsible for aneurysm development [16].Two major categories of gene alterations have been established, including mutations in transforming growth factor beta (TGF-β) signaling cascade components, also known as TGF-β vasculopathies.The second set of genetic mutations includes various components of the smooth muscle contractile apparatus [3].Approximately 30 genes involved in the development of syndromic or non-syndromic TAA development have been established, with many causative mutations remaining unidentified [16].
The PR domain is a protein-protein interaction of roughly 100 amino acids, and PRdomain-containing proteins are often involved in transcriptional regulation [17].Several PR-domain-containing proteins have been linked with cell differentiation and tumorigenesis [18].The PRDM5 gene is located on chromosome 4q27 and is a 23-exon protein coding gene.The protein encoded by the PRDM5 gene is a transcription factor of the PR-domain protein family, and contains a PR-domain as well as multiple zinc finger motifs (Figure 3 Following the sequencing of all 35 genes in the TAAD panel, PRDM5 was the only gene demonstrating a genetic variation in these cases, making this report the first to associate the clinical phenotype of aortopathy and single nucleotide variants in PRDM5.We cannot attribute causation, as this requires further detailed characterizations of these SNV regarding protein structure, resultant biochemistry, and demonstration of the tissue level effect.However, we anticipate that our work will generate interest in these mechanistic studies to demonstrate causal effects between genetic variants in PRDM5 on tissue changes within the extra cellular matrix that subsequently lead to aortopathy.

Discussion: PRDM5
The identification of genetic variants that are responsible for the hereditary development of TAAs is necessary for establishing molecular mechanisms that are responsible for aneurysm development [16].Two major categories of gene alterations have been established, including mutations in transforming growth factor beta (TGF-β) signaling cascade components, also known as TGF-β vasculopathies.The second set of genetic mutations includes various components of the smooth muscle contractile apparatus [3].Approximately 30 genes involved in the development of syndromic or non-syndromic TAA development have been established, with many causative mutations remaining unidentified [16].
The PR domain is a protein-protein interaction of roughly 100 amino acids, and PRdomain-containing proteins are often involved in transcriptional regulation [17].Several PR-domain-containing proteins have been linked with cell differentiation and tumorigenesis [18].The PRDM5 gene is located on chromosome 4q27 and is a 23-exon protein coding gene.The protein encoded by the PRDM5 gene is a transcription factor of the PR-domain protein family, and contains a PR-domain as well as multiple zinc finger motifs (Figure 3  The PRDM5 protein was found to have a variety of functions as a transcription factor, being involved as a tumor suppressor gene in epithelial cancer and as a regulator of extracellular matrix development in corneal and bone cells [20].The PRDM5 transcription factor is critical for extracellular matrix (ECM) development and maintenance in the cornea.The downregulation of genes encoding fibrillar collagens (e.g., COL4A1 and COL11A1), connective tissue components (e.g., HAPLN1), and molecules that regulate cellular migratory patterns and adhesion (e.g., EDIL3 and TGFB2) was found in patients with PRDM5 mutations on a microarray analysis of dermal fibroblasts.PRDM5 mutations resulted in significant corneal fragility, and the central corneal thickness was markedly The PRDM5 protein was found to have a variety of functions as a transcription factor, being involved as a tumor suppressor gene in epithelial cancer and as a regulator of extracellular matrix development in corneal and bone cells [20].The PRDM5 transcription factor is critical for extracellular matrix (ECM) development and maintenance in the cornea.The downregulation of genes encoding fibrillar collagens (e.g., COL4A1 and COL11A1), connective tissue components (e.g., HAPLN1), and molecules that regulate cellular migratory patterns and adhesion (e.g., EDIL3 and TGFB2) was found in patients with PRDM5 mutations on a microarray analysis of dermal fibroblasts.PRDM5 mutations resulted in significant corneal fragility, and the central corneal thickness was markedly decreased [21].We hypothesize that if a genetic variation in PRDM5 encodes for an aberrant protein, a sub-sequent disruption in these protein functions (Figure 4) will lead to aneurysm development.Our laboratory has begun further exploration to explore these underlying mechanisms.
Life 2023, 13, x FOR PEER REVIEW 6 of 8 decreased [21].We hypothesize that if a genetic variation in PRDM5 encodes for an aberrant protein, a subsequent disruption in these protein functions (Figure 4) will lead to aneurysm development.Our laboratory has begun further exploration to explore these underlying mechanisms.PRDM5 mutations in human beings are associated with the development of brittle cornea syndrome (BCS).BCS is an autosomal recessive disorder of the connective tissue that is characterized by both ocular and extra-ocular features.Notable phenotypic features including blue sclera, keratoconus, deafness, and joint hypermobility are present, with the suggested mechanism being a result of PRDM5′s role in extracellular matrix homeostasis.Further strengthening this linkage are the causal relationship between BCS and genetic mutations in another ECM regulating protein, ZNF469 [21,22], and our group's recent reports associating variants in this gene with aortic and arterial aneurysmal and dissection disorders [23,24].The disruption of the ECM via mutations in Exon 3 and Exon 4 of PRDM5 supports our hypothesis that these variations may contribute to a loss of vascular integrity, leading to subsequent aneurysm formation (Figure 5).PRDM5 mutations in human beings are associated with the development of brittle cornea syndrome (BCS).BCS is an autosomal recessive disorder of the connective tissue that is characterized by both ocular and extra-ocular features.Notable phenotypic features including blue sclera, keratoconus, deafness, and joint hypermobility are present, with the suggested mechanism being a result of PRDM5 s role in extracellular matrix homeostasis.Further strengthening this linkage are the causal relationship between BCS and genetic mutations in another ECM regulating protein, ZNF469 [21,22], and our group's recent reports associating variants in this gene with aortic and arterial aneurysmal and dissection disorders [23,24].The disruption of the ECM via mutations in Exon 3 and Exon 4 of PRDM5 supports our hypothesis that these variations may contribute to a loss of vascular integrity, leading to subsequent aneurysm formation (Figure 5).
Limitations of our study include the recognition that although the variants described in our report are missense mutations, we suspect that they are likely to encode for truncated proteins.Further, though it is unknown whether the variants described are in the cis or trans position, our expectation with these phenotypes is the trans position, thereby more likely coding for proteins with aberrant structure.The variants identified in the report were not present in gnomAD, and this significantly reduces the possibility of these being "cold" variants.We believe that further detailed mechanistic study will help to better define the role of the mutations we have discovered in subsequent protein biochemical function and the resultant phenotypic effects.An arginine-to-histidine substitution at codon 83 on Exon 3 was seen in Case 1, and a glutamic-acidto-alanine substitution at codon 129 on Exon 4 was present in Case 2. We hypothesize that these substitutions led to a loss of function of the PRDM5 protein, which affects ECM development and maintenance in our subjects, subsequently leading to aneurysm development.
Limitations of our study include the recognition that although the variants described in our report are missense mutations, we suspect that they are likely to encode for truncated proteins.Further, though it is unknown whether the variants described are in the cis or trans position, our expectation with these phenotypes is the trans position, thereby more likely coding for proteins with aberrant structure.The variants identified in the report were not present in gnomAD, and this significantly reduces the possibility of these being "cold" variants.We believe that further detailed mechanistic study will help to better define the role of the mutations we have discovered in subsequent protein biochemical function and the resultant phenotypic effects.

Conclusions
TAAs are clinically silent pathologies with potentially fatal complications, and an increasing amount of novel pathologic mutations are being discovered with further research.This report of two subjects is the first to make an association between PRDM5 and TAA development and contributes to the evolving understanding of how mutations affecting extracellular-matrix-encoding genes may lead to vascular aneurysms.Further research establishing the pathogenicity of PDRM5 mutations in aneurysm formation will advance screening and care for patients with this disease state.
Funding: This research was supported by the Potishman Foundation through a generalized research restricted grant to the Sathyamoorthy Laboratory at the Burnett School of Medicine at TCU. Funding number: Potishman Foundation: 02 Institutional Review Board Statement: Ethical review and approval were waived for this study per TCU IRB guidelines due to the number of participants and their individual willingness to provide fully informed written consent for this report.These consents are on file at CCMS-FW.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.Written informed consent has been obtained from the patient(s) to publish this paper.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.The data are not publicly available due to patient privacy considerations.

Acknowledgments:
The authors wish to acknowledge staff at CCMS-FW for helping coordinate patient visits and consents, and Ambry Genetics for clinical genetic testing.An arginine-to-histidine substitution at codon 83 on Exon 3 was seen in Case 1, and a glutamic-acidto-alanine substitution at codon 129 on Exon 4 was present in Case 2. We hypothesize that these substitutions led to a loss of function of the PRDM5 protein, which affects ECM development and maintenance in our subjects, subsequently leading to aneurysm development.

Conclusions
TAAs are clinically silent pathologies with potentially fatal complications, and an increasing amount of novel pathologic mutations are being discovered with further research.This report of two subjects is the first to make an association between PRDM5 and TAA development and contributes to the evolving understanding of how mutations affecting extracellular-matrix-encoding genes may lead to vascular aneurysms.Further research establishing the pathogenicity of PDRM5 mutations in aneurysm formation will advance screening and care for patients with this disease state.

Figure 3 .
Figure 3. Simplified structure of the PRDM5 gene.The gene, located on chromosome 4q27, is roughly 100 amino acids and contains 23 exons.Notable characteristics include a PR domain and multiple zinc finger motifs.PR-domain-containing proteins are frequently involved in transcriptional regulation.

Figure 3 .
Figure 3. Simplified structure of the PRDM5 gene.The gene, located on chromosome 4q27, is roughly 100 amino acids and contains 23 exons.Notable characteristics include a PR domain and multiple zinc finger motifs.PR-domain-containing proteins are frequently involved in transcriptional regulation.

Figure 4 .
Figure 4.The PRDM5 protein is responsible for ECM development and maintenance through downstream effects on various proteins and molecules.PRDM5 plays a role in the regulation of fibrillar collagens such as COL4A1 and COL11A1, connective tissue components including HAPLN1, and cell migration and adhesion molecules such as EDIL3 and TGFB2.Downregulation of these downstream proteins and molecules in patients with PRDM5 mutations is potentially responsible for ECM dysregulation and subsequent aneurysm formation.

Figure 4 .
Figure 4.The PRDM5 protein is responsible for ECM development and maintenance through downstream effects on various proteins and molecules.PRDM5 plays a role in the regulation of fibrillar collagens such as COL4A1 and COL11A1, connective tissue components including HAPLN1, and cell migration and adhesion molecules such as EDIL3 and TGFB2.Downregulation of these downstream proteins and molecules in patients with PRDM5 mutations is potentially responsible for ECM dysregulation and subsequent aneurysm formation.

Figure 5 .
Figure 5. Mutations in protein-coding Exon 3 and Exon 4 of PRDM5 were present in our subjects.An arginine-to-histidine substitution at codon 83 on Exon 3 was seen in Case 1, and a glutamic-acidto-alanine substitution at codon 129 on Exon 4 was present in Case 2. We hypothesize that these substitutions led to a loss of function of the PRDM5 protein, which affects ECM development and maintenance in our subjects, subsequently leading to aneurysm development.

Figure 5 .
Figure 5. Mutations in protein-coding Exon 3 and Exon 4 of PRDM5 were present in our subjects.An arginine-to-histidine substitution at codon 83 on Exon 3 was seen in Case 1, and a glutamic-acidto-alanine substitution at codon 129 on Exon 4 was present in Case 2. We hypothesize that these substitutions led to a loss of function of the PRDM5 protein, which affects ECM development and maintenance in our subjects, subsequently leading to aneurysm development.