INVENTORY OF SUPPLEMENTARY DATA
Case reports
Supplementary Table S1
Supplementary Table S2
Supplementary Table S3
Supplementary Figure S1
Supplementary Figure S2
Case Reports
Family 1
The male proband (1-III-1) was evaluated up to the age of 5 years because of tentative diagnosis of Marfan syndrome but subsequently lost for follow-up. He presented with facial and skeletal features including malar hypoplasia, high arched palate, pectuscarinatum, reduced elbow extension, long fingers, pesplanum and joint hypermobility. Upon initial presentation, at the age of 26 months, he had a height of 97 cm (99th percentile) and weight of 15 kg (90th percentile). He had an aortic root size of 21 mm, corresponding with a Z-score of 3 and normal ascending aortic diameters. He also presented with mild learning problems. His family history showed that his mother (1-II-2) was known with a dilated aorta and died suddenly at age 36 yrs. No autopsy was performed. The maternal grandmother (1-I-2) had an aortic dissection at age 56 years and died couple months after initial cardiovascular surgery. A brother of the mother (1-II-3 is also followed for dilated aorta but without further available details.
Family 2
The male proband (2-III-1) presented at the age of 18 years old with possible diagnosis of Marfan syndrome. His height is 189 cm for weight of 61.7 kg. He had the following systemic findings: striaedistensiae, pectusexcavatum (with surgery at age 16), positive wrist and thumb sign, reduced extension of the elbow, pesplanum and joint hypermobility. Facial features included high arch palate and dolichocephaly. His cardiovascular exam is significant for mitral valve prolapse, pulmonary artery aneurysm, aortic root aneurysm (37 mm, Z-score 3) and normal ascending aortic diameters. His family history is significant for some marfanoid skeletal features in his sister (2-III-1) and aortic root echocardiographic dimensions at the upper limits of normal in the mother (2-II-2). No ectopialentis was reported in the family. The maternal grandmother (2-I-2) died at age 50 yrs, but this was stated to be unrelated to cardiovascular problems.
Family 3
The male proband (3-III-1) collapsed at age 15 years due to thoracic aortic dissection. The last available echocardiographic exam 19 days earlier demonstrated a dilated aortic root size at the sinus of valsalva of 45 mm, for which he was on Losartan. He had a small secundum atrial septal defect. His facial features are significant for frontal bossing, a broad nasal root, prominent eyes, hypertelorism, downslanting palpebral fissures. Examination of the limbs showed short fingers with broad thumbs, generalized joint hypermobility except for contractures of the distal interphalangeal joints of both third fingers, lateral deviation of the second toes with overriding of the third toe. Other relevant examinations include mild dilatation of third and lateral ventricle on brain MRI at age 5 years.
Radiograph of both hands at the age of 10 yrs (3-III-1) reveals an asymmetry in the size of the carpal bones with the ones on the right side being larger. Overall, there is symmetric shortening and broadening of the metacarpals and phalanges that appear osteopenic with relatively thin cortices. This is most pronounced for the middle phalanges that show a configuration resembling Babushka dolls. Lateral view of the spine demonstrates platyspondyly with at the thoracolumbar transition mild anterior tongueing. The vertebral bodies are most flattened in their posterior portion. Anteroposterior view of the lower limbs at the age of 10 years 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 a S-shaped configuration of the diaphysis (the latter also present for the fibula). The distal tibial epiphysis is dysplastic.
His brother (3-III-2) has similar facial features with relatively short stature (0.4th percentile) and relative macrocephaly (91th-98th percentile). He presents also with broad thumbs, contractures of proximal and distal interphalangeal joints of the fingers but otherwise generalized joint hypermobility and a very soft skin. He had his aortic root replaced at age 13 years with a root diameter of 34 mm. Both brothers also had hypertrichosis, affecting the back and legs. Both boys had normal intelligence.
Further family history is significant for consecutive normal echocardiographies in their mother (3-II-2) (at ages 40 and 44 yrs). She appears facially similar. She is of average height with no joint hypermobility. The mother has two brothers who both died at young age. The eldest brother (3-II-3) died at age 19 yrs old due to thoracic aortic dissection. His clinical exam showed height of 172.5 cm, frontal bossing, proptosis, joint hypermobility, long fingers and flat feet. The other brother (3-II-4) died at age 17 yrs due to cervical spine compression after vertebral subluxation. Autopsy report mentions a reduction in the size of the foramen magnum, protrusion of the atlas and axis bones with severe narrowing of the dural canal and compression of the spinal cord. The pathologist also described an asymmetry of the skull bones with abnormal shape of the mid-line bones resulting in narrowing of the span of the sphenoidal wings and enlargement of the sellaturcica. The brain autopsy showed mild enlargement of the cerebral ventricles with congestion of the vasculature. The ascending aorta was mildly enlarged. He measured 150 cm and weighed 60 kg. He was known with frontal bossing, hypertelorism, joint laxity and rather long fingers. He had bilateral flat feet with overriding second and fourth toes on the right foot and broad first toes. His medical history includes surgery because of bilateral hip dislocation.
The maternal grandparents (3-I-1 and 3-I-2) are still alive and have both had normal echocardiograms in the past.
Family 4
The male proband (4-II-1) presented in the neonatal period with hypertelorism, downslanting palpebral fissures, bifid uvula, umbilical hernia and marked joint hypermobility with dislocated hips. He has spatulous appearance of the fingers. Other striking observation was the presence of gingival hypertrophy with delayed tooth eruption. He was diagnosed with aortic root aneurysm (Z-score 6.4), dilatation of the ascending aorta (Z-score 5.9) and aneurysm of the patent ductusarteriosus. The latter showed spontaneous thrombosis and involution. Cardiac MRI also demonstrated aortic and arterial tortuosity. Skeletal radiographs at the age of 1.5 years revealed bilateral hip dislocation (treated conservatively), broadened and triangle shaped middle phalanges, broad terminal phalanges and a broad thorax. At age 1.5 years, his height is 72 cm (< 3rd percentile), weight 8.5 kg (<3rd percentile) and occipito-frontal circumference 50.6 cm (95th percentile) (relative macrocephaly). At 2 years, he measured 77 cm (< 3rd percentile). Prenatal and postnatal brain ultrasound revealed dilated lateral ventricles (left greater than right).
His mother (4-I-2) was evaluated at age 30 yrs. She measures 168 cm for weight of 65 kg. She presents hypertelorism, downslanting palpebral fissures, joint hypermobility with shoulder luxation in childhood, mild scoliosis, pesplani, easy bruising, striae and delayed wound healing. She has aortic root dilatation (41 mm, Z-score 4,3) and mild aortic valve regurgitation. Interestingly, she had three surgeries in childhood because of gingival hypertrophy.
Family 5
The male proband (5-II-1) presented at age 35 years with an aortic root aneurysm of 50 mm (Z-score 5.4) and an ascending aorta of 35 mm. Because of subsequent larger measurements (up to 55 mm) on MRA, he underwent valve-sparing aortic root replacement and repair of his aortic valve. Sections from his aorta showed areas with severe medial degeneration and some areas are also associated with moderate atherosclerosis. His clinical examination showed no hypertelorism, mild malar hypoplasia and dolichocephaly. There was no joint hypermobility (Beighton 0/9), pectus deformity or scoliosis. He did have striae with otherwise normal skin. His uvula was broad, but not bifid. His height was 173.2 cm for a weight of 113 kg. His brain and neck MRA were normal.
His mother (5-I-2; 66 years old) was subsequently found to have aortic root dilatation (39 mm, corresponding to Z-score for her BSA of 1.7 (height of 153.9 cm and weight 99.1 kg) or Z-score = 5,6 for height only. She had a prior history of hypertension, hypercholesterolemia and obesity. Her medical history was significant for brain aneurysms surgery at age 31 years, diagnosed after an episode of severe headache. During the surgical repair a second brain aneurysm was uncovered. Her last brain imaging was performed at age 32 years. She had three pregnancies of which she lost one (5-II-2) at 26 weeks due to preterm labour. She did have pregnancy related striae and mentioned joint hypermobility as a child. Physical examination demonstrated prominent eyes with hypertelorism (interpupillary distance 65 mm), a normal uvula, no pectus deformity, rather short fingers, striae and varicose veins, Beighton was score 0/9.
The mother of the proband was adopted and therefore had limited information about her biological relatives. She was aware that two of her paternal uncles died in their fourties because of unspecified cardiac causes.
Table S1 BGN primers used for PCR and Sanger Sequencing
Exon / Forward Primer / Reverse Primer2 / 5´-CTGGCATGGAGAAGACAGGTGG-3´ / 5´-CACAAGTGGGCACAAACTGC-3´
3 / 5´-TGGCTCCAAGTTCATGCTGG-3´ / 5´-ACGAGTCCCCGAACCCCT-3´
4 / 5´-GTGTGCGTGTGAGGAAACTG-3´ / 5´-CATACTCTTTCTTGAACTGAGCC-3´
5 and 6 / 5´-CAGAACTGCTTTCAGGAGAC-3´ / 5´-GGAGAATGACACGGACTCAA-3´
7 / 5´-AGAGTCCCTCAGTGCTGTG-3´ / 5´-TCTGCCACATCTTTCTGGTGT-3´
8 / 5´-GCAGAGGCAACAGCAAAATGC-3´ / 5´-CCCCATCAGGATGTCTGGC-3´
PCR protocol for all amplicons: Touchdown PCR from 65ᵒC to 55ᵒC.
Table S2 Nucleotide and protein prediction programs
Family / DNA change / Protein change / MutationTaster / Sift / PolyPhen2 / ConSurf / MaxEnt
Scan / NNSPLICE / HSF
2 / c.908A>C / p.Gln303Pro / Disease causing (1.000) / Damaging (0.001) / Probably Damaging (1.000) / Conserved amino acid (8/9) / -5.4% / -0.2% / -4.9%
5 / c.238G>A / p.Gly80Ser / Disease causing (1.000) / Damaging (0.029) / Probably Damaging (0.985) / Conserved amino acid (7/9) / -100% / -98.1% / -4.9%
Alamut Visual version 2.7.1. was used for splice site predictions of MaxEntScan, NNSPLICE, and human splicing finder (HSF).
Table S3 BGN mutation overview
Family / DNA change / Protein change / Known frequency / Mutation type / Proposed mechanism1 / c.5G>A / p.Trp2* / Novel / Nonsense / NMD, LOF
2 / c.908A>C / p.Gln303Pro / Novel / Missense/Splicing / LOF
3 / 21 kb deletion: chrX:152767424-152787984 / / / NA / Deletion / LOF
4 / 28 kbdeletion: chrX:152768438-152795976 / / / NA / Deletion / LOF
5 / c.238G>A / p.Gly80Ser / Novel / Missense/Splicing / NMD, LOF
Frequency was checked in the following databases: Exac, 1000 genomes and Exome variant server.
Figure S1 Fluorescent decorin stainings.
(a-c) Decorin staining of the control sample (a), proband 3-III-2 (b) and proband 5-II-1 (c). Decorin is diffusely expressed in the control sample, while a focal expression of decorin can be observed in proband 3-III-2 and proband 5-II-1. Scale bar indicates 25 µm.
Figure S2 Box plots of pSMAD2 positive nuclei count.
(a) Box plot of the percentage of pSMAD2 positive nuclei on the total number of nuclei in the media. In the media, the percentage of pSMAD2 positive nuclei in the control sample is close to zero. In individuals with a BGN mutation we observe an increase in the percentage of pSMAD2 positive nuclei. This increase is even more pronounced in the LDS probands.
(b) Box plot of the percentage of pSMAD2 positive nuclei on the total number of nuclei in the adventitia. The increase of pSMAD2 positive nuclei in BGN and LDS probands is more pronounced compared to the media.
Supplementary references
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