BMP9 Mutations Cause a Vascular-Anomaly Syndrome with Phenotypic Overlap with Hereditary Hemorrhagic Telangiectasia

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Whitney L. Wooderchak-Donahue,1 Jamie McDonald,2 Brendan O’Fallon,1 Paul D. Upton,3 Wei Li,3 Beth L. Roman,4 Sarah Young,4 Parker Plant,1 Gyula T. Fu¨lo¨p,5,6 Carmen Langa,5,6 Nicholas W. Morrell,3 Luisa M. Botella,5,6 Carmelo Bernabeu,5,6 David A. Stevenson,7 James R. Runo,8 and Pinar Bayrak-Toydemir1,2,*

Hereditary hemorrhagic telangiectasia (HHT), the most common inherited vascular disorder, is caused by mutations in genes involved in the transforming growth factor beta (TGF-b) signaling pathway (ENG, ACVRL1, and SMAD4). Yet, approximately 15% of individuals with clinical features of HHT do not have mutations in these genes, suggesting that there are undiscovered mutations in other genes for HHT and possibly vascular disorders with overlapping phenotypes. The genetic etiology for 191 unrelated individuals clinically suspected to have HHT was investigated with the use of exome and Sanger sequencing; these individuals had no mutations in ENG, ACVRL1, and SMAD4. Mutations in BMP9 (also known as GDF2) were identified in three unrelated probands. These three individuals had epistaxis and dermal lesions that were described as telangiectases but whose location and appearance resembled lesions described in some individuals with RASA1-related disorders (capillary malformation-arteriovenous malformation syndrome). Analyses of the variant proteins suggested that mutations negatively affect protein processing and/or function, and a bmp9-deficient zebrafish model demonstrated that BMP9 is involved in angiogenesis. These data confirm a genetic cause of a vascular-anomaly syndrome that has phenotypic overlap with HHT.

Hereditary hemorrhagic telangiectasia (HHT [MIM Dermal telangiectases and cerebral AVMs are also fea- 187300 and 600376]) is an autosomal-dominantly tures of capillary-malformation (CM)-AVM syndrome inherited vascular-malformation syndrome characterized (CM-AVM [MIM 608354]), caused by mutations in RASA1 by telangiectases and arteriovenous malformations (MIM 139150).7–10 Recurrent nosebleeds have not been (AVMs) and has an incidence of 1 in 10,000 individ- described, and the typical dermal telangiectases generally uals.1 Hallmark features are recurrent epistaxis due to differ from HHT in location and appearance. The telangiec- telangiectases of the nasal mucosa; telangiectases on the tases seen in CM-AVM include both the small punctate lips, hands and oral mucosa; solid-organ AVMs, parti- lesions that characterize HHT and the larger telangiectases cularly of the lungs, liver, and brain; and a family history referred to as CMs. The punctate telangiectases in CM- of the same. Presentation with three of these criteria AVM are more commonly diffuse and tend to cluster in a is considered diagnostic for HHT.2 The dermal telangiecta- region of the trunk or limbs (M. Vikkula, personal commu- ses are typically pinpoint to pinhead sized, very specif- nication). ically concentrated on the hands, face and lips, and not We first performed exome sequencing in 38 unrelated diffuse. Telangiectases on the limbs and trunk are not individuals reported to have HHT2 (by physicians who characteristic. had ordered HHT genetic testing) but in whom no muta- Currently, all known genetic defects that cause HHT are tion had been identified in ENG, ACVRL1 or SMAD4. found within the transforming growth factor beta (TGF-b) Informed consent was obtained from all individuals signaling pathway. Mutations in endoglin (ENG [MIM (approved by the institutional review board [00028740] 131195]), activin A receptor type II-like 1 (ACVRL1, also at the University of Utah). Samples were enriched with known as ALK1 [MIM 601284]), and SMAD4 (MIM the use of exome-targeted biotinylated RNA baits (SureSe- 600993) cause HHT type 1, HHT type 2, and the combined lect 50 Mb) and sequenced with 23 100 bp paired-end juvenile polyposis (JP) and HHT (JP-HHT) syndrome, reads (HiSeq2000, Illumina). The Burrows-Wheeler Aligner respectively.3–5 Approximately 15% of individuals identi- (0.5.9)11 and the Genome Analysis Toolkit (v.1.6)12 were fied clinically as having HHT currently have no known ge- used for data analysis. To reduce the number of false-posi- netic cause,6 suggesting that there are undiscovered genes tive variant calls, we used raw variant sets as input to a associated with HHT. Variant Quality Score Recalibration procedure, and we

1Associated Regional and University Pathologists Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, USA; 2Department of Pathol- ogy, University of Utah, Salt Lake City, UT 84108, USA; 3Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke’s Hospital, Cambridge CB2 0QQ, UK; 4Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA; 5Centro de Investigaciones Biolo´gicas, Consejo Superior de Investigaciones Cientı´ﬁcas, Madrid 28040, Spain; 6Centro de Investigacio´n Biome´dica en Red de Enfermedades Raras, Madrid 28040, Spain; 7Division of Medical Genetics, Department of Pediatrics, University of Utah, Salt Lake City, UT 84108, USA; 8Department of Med- icine, University of Wisconsin School of Medicine and Public Health, Madison, WI 53792, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.ajhg.2013.07.004. Ó2013 by The American Society of Human Genetics. All rights reserved.

530 The American Journal of Human Genetics 93, 530–537, September 5, 2013 Table 1. BMP9 Variants and Case Phenotypes

Nucleotide Protein Presence in Family Individual Clinical Description Change Change Populationa Segregation Variant Classiﬁcation

1 epistaxis, telangiectasia, ﬁrst-degree relative c.254C>T p.Pro85Leu absent NA pathogenic reported to have HHT

2 epistaxis, telangiectasia or CM (back, c.203G>T p.Arg68Leu absent yesb pathogenic shoulders, mouth, face), AVM (thumb), parent and sibling with history of epistaxis

3 epistaxis, telangiectasia (80–100 on the left c.997C>Tc p.Arg333Trpc 0.004 NA suspected to be pathogenic arm and shoulder, 2 on the right palm, 1 possible on the lip); a parent reports frequent epistaxis as a child and teen

4 CM and hemihypertrophy c.51C>Ac 50 UTRc absent NA suspected to be benign

All nucleotide changes were heterozygous. BMP9 RefSeq accession number NM_016204 was used. ‘‘NA’’ stands for not available. aPresence in population refers to the presence of the variant in 1000 Genomes, dbSNP, or 5,400 control exomes. bFather and sister with epistaxis also carry the mutation. cIdentiﬁed by Sanger sequencing.

discarded resulting variants with VQRLOD scores less than sions suggested a cutaneous phenotype that differs from 10.0. classic HHT and has some resemblance to lesions described We prioritized variants by subjecting them to a two-part in individuals with RASA1-related disorders (CM-AVM). classification procedure. First, we computed SIFT,13 Poly- Therefore, BMP9 exons were also Sanger sequenced for 60 Phen-2,14 MutationTaster,15 GERPþþ,16 PhyloP,17 LRT,18 unrelated individuals who had been clinically suspected and SiPhy19 scores for all nonsynonymous variants. We of having a mutation in RASA1 by their referring physician computed scores by consulting a precomputed database but in whom no mutation was identified in this gene. One of all possible scores distributed with the dbNSFP package, variant not present in publically available databases was version 2.0.20 We combined these scores into a single value identified in the BMP9 50 UTR region (c.51C>A) in one by using a linear weighting scheme, and this value re- individual (Table 1), who had macrocephaly, extensive flected the overall damage to the gene in question. Next, CMs, and hemihypertrophy. Given that the individual’s we employed phenotypic ranking by using the VarRanker unaffected mother had the same 50 UTR variant, it is not algorithm (B.O., unpublished data). likely that this variant causes the individual’s phenotype. Pathogenic missense variants were identified in BMP9 Individual 1 (c.254C>T [p.Pro85Leu]) is a 33-year-old (MIM 605120) in 2 of the 38 samples: c.254C>T (p.Pro85- female whose onset of epistaxis occurred in early child- Leu) and c.203G>T (p.Arg68Leu) (Figures S1A and S1B, hood. She reports that her nosebleeds were the most signif- available online, and Table 1) (RefSeq accession number icant during childhood—they occurred two to six times a NM_016204.1). Neither BMP9 variant was present in week—but were still significant enough in her twenties publically available databases, the affected residues were to warrant treatment by nasal cautery. She reported that highly conserved (from humans to zebrafish), and their she currently has approximately three nosebleeds a week corresponding mutations were predicted to be damaging but that they are minor in duration relative to how they by computing algorithms (Table S1).13–15 were during childhood. She had several cutaneous telangi- Next, BMP9 exons were Sanger sequenced in an addi- ectases on her chest and face. No solid-organ AVMs were tional 153 unrelated individuals who had been referred known, but she had not been screened for solid-organ for molecular genetic testing as a result of suspicion of AVMs. Her father died suddenly at the age of 45 years, HHT by a referring physician, although most (86%) met and according to the family, HHT was suspected on the ba- two of the established diagnostic criteria for HHT and the sis of autopsy findings. Unfortunately, this report was not other 14% met three diagnostic criteria. Another variant available to confirm details, but it was the suggestion of (c.997C>T [p.Arg333Trp]) was identified in one individual HHT in her father years ago that led this individual to within this group (Figure S1C and Table 1). Although seek consultation regarding her epistaxis and cutaneous this variant has been previously reported in dbSNP lesions. A sibling had recurrent spontaneous nosebleeds (rs35129734), it is potentially disease causing because it (approximately two per week) and a stroke of unknown eti- is very rare (variant frequency of 0.004), the affected resi- ology at the age of 43 years. No family members were avail- due is strictly conserved among higher vertebrates, and able for examination or variant analysis. the variant is predicted to be deleterious by SIFT,13 Poly- Individual 2 (c.203G>T [p.Arg68Leu]) is a 37-year-old Phen-2,14 and MutationTaster.15 female whose onset of epistaxis occurred at approximately All three individuals identified with a BMP9 mutation 30 years of age. The frequency of her nosebleeds varied had dermal lesions described as telangiectases, yet a from daily to once every 1–2 weeks. The individual had detailed review of the location and appearance of their le- 15–20 dermal vascular malformations on her face, mouth,

The American Journal of Human Genetics 93, 530–537, September 5, 2013 531 Individual 3 (c.997C>T [p.Arg333Trp]) is a 14-year-old male whose onset of epistaxis occurred at approximately 3 years of age. Frequency of epistaxis had ranged from approximately once per week to several times per day. He had 80–100 telangiectases on his left upper extremity from wrist to shoulder, two telangiectases on the palm of his right hand, and one possible telangiectasia on his lower lip. The pathology report of a biopsy of the lesions confirmed them to be telangiectases. No solid-organ involvement (AVM) was known, but he was not screened by the referring physician. Per family history report, his father had nosebleeds of more than once per week in elementary school until his early teens, but none in recent years. DNA was not available from the father for variant analysis. To characterize the functional effects of the three BMP9 variants identified, we evaluated BMP9 levels and processing into the mature, active form of the protein21 (Figure 2A). Comparison of plasmid-transfected human embryonic kidney (HEK) EBNA cell lysates and supernatants by immunoblot using an antibody specific to mature BMP9 revealed that the processing of the p.Arg68Leu and p.Arg333Trp variants to mature BMP9 was far less efficient than that of the wild-type (WT) BMP9, whereas processing of the p.Pro85Leu variant was less impaired (Figure 2B). This reduction of mature BMP9 in the supernatants was later confirmed with a specific ELISA in HEK cells (Figure 2C), as well as in the hepatoma cell line HepG2 (Figure S2). Importantly, control culture supernatants from HepG2 cells showed only low background levels of BMP9 (<10 pg/ml) (data not shown). Thus, all three variants altered BMP9 processing in vitro. Unfortunately, Figure 1. Cutaneous Vascular Lesions in Individual 2 serum samples were not available for testing BMP9 levels Shown are the middle digit of the left hand (A), the dorsal aspect of the right hand (B), several of approximately 20 lesions on the back in these individuals. and shoulders (C), and a vascular lesion previously treated by laser To assess the relative activities of the WT and variant ablation on the right jawline (D). forms of BMP9, we examined their ability to induce BRE- luciferase activity in C2C12 cells transfected with human upper chest, upper mid back, and hands, and they were ALK1 (Figures 2D–2F). Assays of these BMP9 variants in first noticed when she was about 30 years old (Figure 1). C2C12 cells transfected with ALK1 revealed that the The individual reported that the larger malformations p.Arg68Leu and p.Pro85Leu variants exhibited less activity had faded from a darker to lighter pink over time. An than did the WT BMP9 at 10 pg/ml (p.Arg68Leu: 79.2% 5 MRI of the brain did not show AVMs. Her father and sister 10.3% of WT; p.Pro85Leu: 82.2% 5 7.6% of WT) and 30 reported infrequent but recurring nosebleeds, but they pg/ml (p.Arg68Leu: 78.9% 5 4.3% of WT; p.Pro85Leu: were not available for clinical examination. DNA was ob- 78.6% 5 7.2% of WT) equivalent dilutions, but the tained from saliva, and the BMP9 c.203G>T (p.Arg68Leu) p.Arg333Trp variant did not (10 pg/ml equivalent: variant was identified in both the father and the sister. 100.92% 5 11.8% of WT; 30 pg/ml equivalent: 99.6% 5 Individual 2 had abnormal liver enzymes and portal hy- 2.1% of WT) (Figure 2F). No luciferase activity was detected pertension. A liver MRI showed findings suggestive of the in the conditioned medium from HEK EBNA cells trans- hepatic vascular findings seen in HHT.In particular, the arte- fected with the empty pCEP4 vector (data not shown). rial phase was slightly heterogeneous, and several smaller We also tested the biological activity of the BMP9 vari- branches of the hepatic artery were prominent and slightly ants by measuring production of alkaline phosphatase in corkscrew in configuration. Several small focal enhancing the ATDC5 mouse chondrogenic cell line. Alkaline phos- nodules were noted on the arterial phase that equilibrated phatase is a widely accepted marker of the signaling in other phases and were not visible on other sequences, pathway induced by different BMPs and by BMP9 in partic- suggesting small focal nodular hyperplasia. However, this ular.22 At the same protein concentration (1 ng/ml), the individual had also been diagnosed with hepatopulmonary p.Arg68Leu, p.Pro85Leu, and p.Arg333Trp BMP9 variants syndrome, and the cause of her hepatic issues is not clear. showed less activity than did the WT protein (Figure 3).

532 The American Journal of Human Genetics 93, 530–537, September 5, 2013 Figure 2. BMP9 Variants Alter Protein Processing and Binding of Ligands to ALK1 (A) BMP9 diagram depicting the alteration locations, prodomain, and mature protein. The BMP9 precursor encodes a signal sequence from amino acids 1–22, a prodomain from amino acids 23–319 (proBMP9), and the mature BMP9 from amino acids 320–429. Secreted BMP9 comprises amino acids 23–429; furin-type proteases cleave its prodomain to generate mature BMP9.21 (B) Plasmids encoding human WT BMP9 or the p.Arg68Leu, p.Pro85Leu, or p.Arg333Trp variants were transfected into HEK EBNA cells. Cell lysates and conditioned media were fractionated with nonreducing SDS-PAGE and immunoblotted for BMP9 with a mouse monoclonal BMP9 antibody (clone #360107, R&D Systems). For cell lysates, equal protein loading was confirmed by subsequent immu- noblotting for a-tubulin and densitometry of the bands normalized to the a-tubulin bands. Supernatants were also fractionated and immunoblotted for BMP9. A parallel gel was run and stained with Coomassie blue. For verification, the Coomassie-stained bands were sequenced. WT and variant BMP9 proteins fractionated as three bands corresponding to the mature BMP9 dimer associated with both prodomains (~125–150 kDa), the mature BMP9 dimer associated with a single prodomain (75 kDa), and mature BMP9 (22 kDa). Mature BMP9 is indicated by an arrow. Densitometry graphs are also shown. (C) BMP9 levels were determined by a specific ELISA (n ¼ 3). The ELISA was developed with a colorimetric substrate comprising 1 mg/ml 4-nitrophenyl phosphate disodium salt hexahydrate in 1 M diethanolamine (pH 9.8) containing 0.5 mM MgCl2. The assay was developed in the dark at room temperature, and the absorbance was measured at 405 nm. Unknown values were extrapolated from the standard curve with a 4-parameter log curve fit. (D) C2C12 cells were transfected with human ALK1 and treated with R&D Systems BMP9 (between 0.1 and 3,000 pg/ml) or WT BMP9 diluted to equivalent activities. Cells exposed to Lipofectamine (‘‘Lipo’’) alone were treated in parallel with R&D Systems BMP9. All cells were cotransfected with BRE-luciferase and pTK-Renilla. BMP9-driven firefly luciferase responses were normalized to Renilla activity. Data (legend continued on next page)

The American Journal of Human Genetics 93, 530–537, September 5, 2013 533 Taken together, these experiments demonstrate that the ingly, Bmp9-knockout mice did not show any vascular three BMP9 variants affect the biological activity of BMP9. phenotype in retinal vascularization, and no abnormal Next, we performed bmp9 knockdown experiments retinal vascularization was noted in the three individuals in zebrafish to identify whether they would exhibit a with BMP9 variants. Furthermore, BMP9 specifically plays vascular phenotype similar to HHT. Tg(kdrl:egfp)la116;Tg a physiologic role in the control of adult blood-vessel (gata1.DsRed2)sd2 zebrafish embryos23,24 were injected at maintenance and angiogenesis by targeting ALK1 on endo- the 1- to 4-cell stage with 7 ng bmp9 translation-blocking thelial cells.28 morpholino (50-GGAGCAAATGTCCTACGCGCCACAT-30) BMP9 exerts its functional effects by binding to specific or standard control morpholino (GeneTools). Compared endothelial cell surface receptors, namely the auxiliary re- to control morphants, bmp9 morphants exhibited small ceptor endoglin and the serine-threonine kinase ALK1, but significant decreases in both anterior-posterior and both members of the highly conserved TGF-b superfamily. dorsal-ventral axes, but trunk and tail anatomy were other- Then, the BMP9-dependent activation of ALK1 leads to the wise normal (Figures 4A and 4B). Vascular patterning in phosphorylation of Smad1, Smad5, and Smad8, and the re- bmp9 morphants was relatively normal, although subtle sulting phospho-Smad proteins associate with Smad4 to defects were detected in maturation of the caudal vein. form a Smad complex that translocates to the nucleus to Although all control morphants exhibited a dominant regulate gene expression in human microvascular endo- ventral-vein return by 2 days postfertilization (dpf) (n ¼ thelial cells. In addition, ALK1 activation by BMP9 further 86/86), in bmp9 morphants the caudal venous plexus increases by overexpression of the coreceptor endoglin.28 failed to resolve and both dorsal and ventral veins Furthermore, BMP9 signaling via endoglin promotes a continued to carry blood flow (n ¼ 76/91, 83.5%; Figures switch from a chemokine-responsive autocrine phenotype 4C and 4D). This phenotype persisted as late as 5 dpf, sug- to a chemokine-nonresponsive paracrine state that re- gesting that it was not the result of developmental delay presses endothelial cell migration and promotes vessel but instead reflected impaired remodeling. Repeating the maturation.29 These data strongly suggest the involvement knockdown of bmp9 with a splice-blocking morpholino re- of BMP9 in a common TGF-b signaling pathway shared by sulted in the same venous remodeling defect (data not ALK1, endoglin, and Smad4. Because mutations in ENG, shown), supporting a role for BMP9 in angiogenesis. In ze- ALK1, and SMAD4 give rise to different types of HHT brafish, alk1 mutations result in robust cranial AVMs.25,26 (HHT1, HHT2, and JP-HHT, respectively), it can be postu- However, we detected no cranial AVMs in bmp9 morphants lated that alterations in BMP9, the upstream component (n ¼ 0/207), suggesting that other ligands such as BMP10 of this signaling route, cause a vascular-malformation syn- might compensate for BMP9 in the development of these drome with phenotypic overlap with HHT (Figure 5). Sup- cranial vessels (B.R., unpublished results). porting this view, many of the mutations described in ENG Overall, three missense variants in BMP9 were identified involve its orphan domain, a region responsible for bind- in 191 individuals with features of HHT. These three amino ing to BMP9.30–33 acids are conserved from humans through zebrafish, pre- We observed that the p.Arg68Leu and p.Arg333Trp vari- dicted to be damaging, and shown by in vitro studies to ants dramatically affected the degree of processing in the alter BMP9 processing to the mature form and BMP9 activ- HEK EBNA expression system and thus reduced the ity to varying degrees (Figures 2 and 3). Furthermore, amount of mature BMP9 that was being produced inside knockdown of bmp9 in zebrafish resulted in defects in the expressing cells. Furthermore, the secretion of the venous remodeling (Figure 4), supporting a role for BMP9 mature and proBMP9 forms of the p.Arg68Leu and in angiogenesis. This is consistent with the reported role p.Arg333Trp variants was dramatically reduced. Intrigu- for BMP signaling during vascular development: blocking ingly, the p.Pro85Leu variant also exhibited reduced levels BMP9 with a neutralizing antibody in newborn mice of the proBMP9 form, but the processing was less impaired significantly increased retinal vascular density.27 Surpris- than in the other two variants. In addition to displaying are the mean 5 SEM (n ¼ 4 wells) for a representative experiment. The activity of the WT protein was titrated in comparison to commercial BMP9 for the establishment of an activity profile. C2C12 cells transfected with ALK1 became sensitive to commercial BMP9 (mature BMP9, amino acids 320–429) at concentrations as low as 1 pg/ml. (E) Supernatants from (B) were diluted so that there were equal amounts of mature BMP9 in the media conditioned with WT and variant BMP9. The activity profiles for WT BMP9 were similar to the commercial BMP9 profile, albeit with reduced activity at the lowest concentrations of 1 and 10 pg/ml. For each variant, relative supernatant volumes necessary for achieving equivalent amounts of the mature WT BMP9 were determined. (F) C2C12 cells were transfected with human ALK1 and treated with WT or variant BMP9. Cells exposed to Lipofectamine alone were treated in parallel with WT BMP9. All cells were cotransfected with BRE-luciferase and pTK-Renilla. BMP9-driven firefly luciferase responses were normalized to Renilla activity. The range over which ALK1 activation occurs is indicated. Data are the mean 5 SEM for n ¼ 4 wells from a representative experiment. Top dilutions were prepared at 1 (WT) to 8.3 (p.Arg68Leu) to 1.3 (p.Pro85Leu) to 4 (p.Arg333Trp), and serial dilutions of each BMP9 variant were prepared with the same dilution series as with commercial BMP9. Assays of these BMP9 variants in C2C12 cells transfected with ALK1 revealed that the p.Arg68Leu and p.Pro85Leu variants exhibited less activity than WT BMP9 at 10 pg/ml (p.Arg68Leu: 79.2% 5 10.3% of WT; p.Pro85Leu: 82.2% 5 7.6% of WT) and 30 pg/ml (p.Arg68Leu: 78.9% 5 4.3% of WT; p.Pro85Leu: 78.6% 5 7.2% of WT) equivalent dilutions, but the p.Arg333Trp variant did not (10 pg/ml equivalent: 100.92% 5 11.8% of WT; 30 pg/ml equivalent: 99.6% 5 2.1% of WT).

534 The American Journal of Human Genetics 93, 530–537, September 5, 2013 of the total number of individuals with suspected HHT) of them were found to have a BMP9 variant. These three individuals all had epistaxis and dermal lesions described as telangiectases; however, the lesions were less concentrated on the hands and mouth than in individuals with mutations in ENG, ACVRL1,orSMAD4. In individual 2, the size and appearance of some lesions were more similar to the ‘‘atyp- ical CM’’ seen in CM-AVM. In individual 3, the lesions were more punctate and resembled those in HHT in appearance, but the number and distribution were more typical of telangiectasia described in CM-AVM. The variants were shown to be present in two symptomatic first-degree relatives of one index case. However, no family members were available for examination by our group. Molecular screening of BMP9 in individuals suspected to have HHT should be considered in individuals with Figure 3. Missense Substitutions in BMP9 Alter Protein Activity The mouse chondrogenic cell line ATDC5 was incubated with epistaxis and small blanching vascular lesions on the 1 ng/ml of WT BMP9 or the p.Arg68Leu, p.Pro85Leu, or upper body and trunk rather than limited to the hands, p.Arg333Trp variants produced in HepG2 cells, as shown in face, and mouth (as is typical of HHT). It is of note that Figure S2. Alkaline phosphatase activity was measured in permea- the individuals in this study, and many with HHT, have bilized ATDC5 cells with the use of p-nitrophenyl phosphate as a soluble substrate. Relative activity units are shown in the vertical relatively infrequent, mild nosebleeds not routinely axis. *p < 0.001; **p < 0.07. reported to physicians during medical history. Because of the small number of individuals presented here and the changes in processing, the p.Arg68Leu and p.Pro85Leu fact that evaluation for solid-organ AVMs was not BMP9 variants exhibited slightly reduced activity with routinely performed, little can be concluded about the ALK1. A reduction in activity is consistent with these mu- presence or absence of internal AVMs in this disorder. tations’ being in a region that might interact with ALK1.21 However, in the one individual in which imaging of solid We did not observe reduced activity of the p.Arg333Trp organs (brain, lungs, and liver) was performed, liver find- variant in our C2C12 activity assay, and we observed ings similar to those seen in HHT were noted. Interestingly, only a slight reduction in activity of the same variant in BMP9 has been reported to be predominantly expressed by ATDC5 cells (Figure 3). This mutation might affect a region the hepatocytes,34 suggesting that the liver vasculature is a that binds to type II or type III receptors, including endo- potential target of BMP9 deficiency. glin. In summary, it is likely that the different BMP9 vari- Continued molecular analysis and clinical description in ants each affect particular aspects of BMP9 processing, families affected by various patterns of CM and/or telangi- secretion, and receptor activity and thus lead to reduced ectases will lead to continued expansion and refinement of functionality of BMP9. hereditary conditions characterized by cutaneous vascular In our cohort of 191 individuals suspected to have HHT lesions. Both will help guide proper surveillance and on examination by a referring physician, 3 (1.6%, or 0.24% possible interventions for this group of individuals.

Figure 4. bmp9 Morphant Zebrafish Display Impaired Venous Remodeling (A and B) Bright-field images of live embryos; boxed areas in (A) and (B) designate regions of focus in (C) and (D), respectively. The scale bar represents 200 mm. (C and D) Two-photon confocal images of circulation in the caudal vein plexus at 2 dpf. Endothelial cells are green, and red blood cells are magenta. The scale bar represents 50 mm. Abbreviations are as follows: DV, dorsal vein; and VV, ventral vein. Zebrafish Tg(kdrl:egfp);Tg(gata1:dsRed2) embryos were injected at the 1- to 4-cell stage with 7 ng control morpholino (A and C) or 7 ng bmp9 morpholino (B and D) and imaged at 2 dpf. Embryos were imaged with a Leica TCS SP5 confocal microscope (Leica Microsystems) outfitted with an APO L 203/1.00 water-immersion objective, standard visible lasers, and Mai Tai DeepSee IR laser (Spectra-Physics, Newport Corp.). EGFP was imaged at 900 nm, and DsRed2 was imaged at 561 nm. Z-series were collected at 2 mm intervals, and two-dimensional projections were generated with MetaMorph 7.7 (Molecular Devices). All images show left anterior lateral views.

The American Journal of Human Genetics 93, 530–537, September 5, 2013 535 Web Resources

The URLs for data presented herein are as follows:

Online Mendelian Inheritance in Man (OMIM), http://www. omim.org RefSeq, http://www.ncbi.nlm.nih.gov/RefSeq

References

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