Loss-of-function mutations in the X-linked biglycan gene cause a severe syndromic form of thoracic aortic aneurysms and dissections

Abstract

Purpose: Thoracic aortic aneurysm and dissection (TAAD) is typically inherited in an autosomal dominant manner, but rare X-linked families have been described. So far, the only known X-linked gene is FLNA, which is associated with the periventricular nodular heterotopia type of Ehlers-Danlos syndrome. However, mutations in this gene explain only a small number of X-linked TAAD families.

Methods: We performed targeted resequencing of 368 candidate genes in a cohort of 11 molecularly unexplained Marfan probands. Subsequently, Sanger sequencing of BGN in 360 male and 155 female molecularly unexplained TAAD probands was performed.

Results: We found five individuals with loss-of-function mutations in BGN encoding the small leucine-rich proteoglycan biglycan. The clinical phenotype is characterized by early-onset aortic aneurysm and dissection. Other recurrent findings include hypertelorism, pectus deformity, joint hypermobility, contractures, and mild skeletal dysplasia. Fluorescent staining revealed an increase in TGF-β signaling, evidenced by an increase in nuclear pSMAD2 in the aortic wall. Our results are in line with those of prior reports demonstrating that Bgn-deficient male BALB/cA mice die from aortic rupture.

Conclusion: In conclusion, BGN gene defects in humans cause an X-linked syndromic form of severe TAAD that is associated with preservation of elastic fibers and increased TGF-β signaling.Genet Med 19 4, 386-395.

Figure 1

Mutation analysis and BGN structure. (a) Pedigrees of the families with their respective mutations. Squares represent males, circles represent females, filled symbols represent aortic aneurysm/dissection and/or systemic involvement, half-filled symbols represent individuals with incomplete clinical data, and + or − sign denotes presence or absence of BGN mutation. The brackets represent an individual who was adopted into the family. (b) Location of BGN on the X chromosome and identified mutations. Deletions are marked with a red bar. ATP2B3 isoform 1 represents ENST00000359149. ATP2B2 isoforms 2 and 3 represent ENST00000370186 and ENST00000349466. (c) Conservation of residues on amino acid position 80 and position 303 in other species and in human DCN.

Figure 2

Aberrant splicing in family 5. In the proband of family 5, three aberrantly spliced mRNAs are produced. The cDNA (converted after inhibition of nonsense-mediated decay with puromycin) was cloned into a vector and the number of colonies with the different products was counted (total counted n = 94).

Figure 3

Clinical features. (a) Clinical features include hypertelorism, malar flattening, down-slanting palpebral fissures (3-II-2/3/4, 3-III-1/2, and 4-II-1), proptosis (3-III-1/2), joint hypermobility (4-II-1), short spatulous fingers (3-III-1/2), and camptodactyly of fingers and toes (3-III-1/2). (b) Skeletal survey of patient 3-III-1 at age 10 years and of patient 4-II-1 at age 1.5 years. Radiograph of both hands (3-III-1) reveals asymmetry in the size of the carpal bones, with the ones on the right being larger. Overall, there is symmetric shortening and broadening of the metacarpals and phalanges that appear osteopenic with relatively thin cortices. Lateral view of the spine (3-III-1) demonstrates platyspondyly with thoracolumbar transition mild anterior tonguing. Anteroposterior view of the lower limbs (3-III-1) shows that the distal (sub)metaphyseal region of the femur is widened and the knee epiphyses are flattened. There is also mild undermodeling of the tibia with an S-shape configuration of the diaphysis. The distal tibial epiphysis is dysplastic. Skeletal radiographs of proband 4-II-1 reveal bilateral hip dislocation (treated conservatively), broadened and triangular middle phalanges, broad terminal phalanges, and a broad thorax.

Figure 4

Histological and fluorescent staining. (a–d) Verhoeff-Van Gieson staining of the control sample (a), proband 3-III-2 (b), proband 5-II-1 (c), and an individual with LDS (d). The elastin fibers in proband 3-III-2 do not show any clear breaks. In the tissue from the individual with LDS, a clear reduction in the elastic fiber content is present. Scale bar indicates 5 mm. (e–h) Trichrome Masson staining of the control sample (e), proband 3-III-2 (f), proband 5-II-1 (g), and an individual with LDS (h). A clear reduction of collagen content is present in proband 3-III-2, whereas an increase is observed in the individual with LDS. (i–l) Biglycan staining of the control sample (i), proband 3-III-2 (j), proband 5-II-1 (k), and an individual with LDS (l). Biglycan is not expressed in proband 3-III-2, whereas in proband 5-II-1 some residual expression of biglycan is present. In the proband with LDS, focal increased expression of biglycan can be observed. Scale bar indicates 25 µm. (m–p) pSMAD2 staining of the control sample (m), proband 3-III-2 (n), proband 5-II-1 (o), and an individual with LDS (p). No pSMAD2-positive nuclei are present in the control sample. In proband 3-III-2, proband 5-II-1, and the proband with LDS, an increase in pSMAD2-positive nuclei can be observed.