LOX Mutations Predispose to Thoracic Aortic Aneurysms and Dissections

Abstract

Rationale: Mutations in several genes have been identified that are responsible for 25% of families with familial thoracic aortic aneurysms and dissections. However, the causative gene remains unknown in 75% of families.

Objectives: To identify the causative mutation in families with autosomal dominant inheritance of thoracic aortic aneurysms and dissections.

Methods and results: Exome sequencing was used to identify the mutation responsible for a large family with thoracic aortic aneurysms and dissections. A heterozygous rare variant, c.839G>T (p.Ser280Arg), was identified in LOX, encoding a lysyl oxidase, that segregated with disease in the family. Sanger and exome sequencing was used to investigate mutations in LOX in an additional 410 probands from unrelated families. Additional LOX rare variants that segregated with disease in families were identified, including c.125G>A (p.Trp42*), c.604G>T (p.Gly202*), c.743C>T (p.Thr248Ile), c.800A>C (p.Gln267Pro), and c.1044T>A (p.Ser348Arg). The altered amino acids cause haploinsufficiency for LOX or are located at a highly conserved LOX catalytic domain, which is relatively invariant in the population. Expression of the LOX variants p.Ser280Arg and p.Ser348Arg resulted in significantly lower lysyl oxidase activity when compared with the wild-type protein. Individuals with LOX variants had fusiform enlargement of the root and ascending thoracic aorta, leading to ascending aortic dissections.

Conclusions: These data, along with previous studies showing that the deficiency of LOX in mice or inhibition of lysyl oxidases in turkeys and rats causes aortic dissections, support the conclusion that rare genetic variants in LOX predispose to thoracic aortic disease.

Keywords: aortic aneurysm; aortic diseases; exome; mutation.

Figure 1. LOX rare variants identified in FTAAD families

A. Pedigrees of families with LOX rare variants. The legend indicates the disease and mutation status of the family members. The age at diagnosis of aortic aneurysm or dissection (“dx”) and age at death (“d”) are shown in years. An asterisk indicates individuals with a bicuspid aortic valve. A dashed circle around a symbol indicates individuals whose DNA was used for exome sequencing. B. Orthologue conservation of LOX variants identified in individuals with TAAD and surrounding amino acids. C. Schematic representation of lysyl oxidase encoded by LOX with the domains of the proteins indicated. The LOX rare variants identified in this study are on the top of the protein diagram and the rare variants identified in European Americans in the NHLBI ESP database are indicated below. Red or blue font designates variants predicted to be possibly or probably damaging, respectively, and black font indicates variants predicted to be benign by PolyPhen-2 analysis.

Figure 2. Aortic pathology associated with aneurysms in individuals with LOX variants

Movat pentachrome and picro sirius red staining was done on control aortic tissue and aortic tissue removed at the time of prophylactic repair of a thoracic aortic aneurysms from patients with the p.Gly202* and p.Ser280Arg mutations. Evidence of medial degeneration is evident by Movat staining in the patients’ aortas as indicated by focal fragmentation of elastic fibers (black) and a decreased number of SMCs (red) when compared to the control aorta. Movat staining also shows the disorganization of elastin deposition in the patients’ aortas compared with control aortas. Picro sirius red staining of collagen fibers shows increased collagen deposition in the patients’ aortas compared with the control aorta.

Figure 3. Missense variants identified in FTAAD patients decrease the enzymatic activity of LOX

A. Immunoblot analyses of HeLa lysates transfected with plasmid containing WT LOX or the individual LOX variants using an antibody directed against HA tag (top) and the LOX protein (middle) show equal expression of a pro-LOX-HA with all plasmids. B. Lysyl oxidase enzymatic activity assays show that LOX activities were significantly decreased when compared with WT for Ser280Arg and Ser348Arg LOX variants, but not for Thr248Ile. All LOX activity data are resulted from two separate transfections, and triplicate analysis of the lysates from each transfection. Lysyl oxidase activities were measured at 30, 60, 90, 120, and 150 minutes, respectively. Data are normalized to activity of the WT LOX with the non-transfected cells activity subtracted. Activity is presented in relative fluorescent units with each data point, error bars indicate standard deviation. *p<0.05 for the comparison of mutant versus WT LOX activity levels.