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Mutation of IGFBP7 Causes Upregulation of BRAF/MEK/ERK Pathway and Familial Retinal Arterial Macroaneurysms

Leen Abu-Safieh,1,10 Emad B. Abboud,2,10 Hisham Alkuraya,1,2,10 Hanan Shamseldin,1 Shamsa Al-Enzi,1 Lama Al-Abdi,1 Mais Hashem,1 Dilek Colak,3 Abdullah Jarallah,4 Hala Ahmad,5 Steve Bobis,5 Georges Nemer,6 Fadi Bitar,7 and Fowzan S. Alkuraya1,8,9,*

Insulin-like growth factor binding proteins (IGFBPs) play important physiological functions through the modulation of IGF signaling as well as IGF-independent mechanisms. Despite the established role of IGFs in development, a similar role for the seven known IGFBPs has not been established in humans. Here, we show that an autosomal-recessive syndrome that consists of progressive retinal arterial macro- aneurysms and supravalvular pulmonic stenosis is caused by mutation of IGFBP7. Consistent with the recently established inhibitory role of IGFBP7 on BRAF signaling, the BRAF/MEK/ERK pathway is upregulated in these patients, which may explain why the cardiac phenotype overlaps with other disorders characterized by germline mutations in this pathway. The retinal phenotype appears to be mediated by a role in vascular endothelium, where IGFBP7 is highly expressed.

Insulin-like growth factors IGF1 (MIM 147440) and IGF2 type in mice in which individual Igfbps have been knocked (MIM 147470) are signaling polypeptides that are involved out.5,7 Therefore, study of IGFBPs in humans has largely in a wide array of physiological functions both embryolog- been limited to their role in cellular metabolism, both ically and postnatally by promoting growth and inhibiting under physiological conditions and in pathologies, mostly apoptosis.1 Their bioavailability to the receptors that malignancies.8–10 mediate their signaling is highly regulated by their binding One of the current authors has previously described a to a family of binding proteins (IGFBPs), seven of which retinal vascular phenotype in which several patients had have been described to date in humans.1 Although IGFBPs a strikingly similar appearance of ‘‘beading’’ along the were initially investigated in connection to IGF signaling, major retinal arterial trunks, with the formation of macro- there is accumulating evidence that they have physiolog- aneurysms. The mode of inheritance could not be estab- ical roles that are independent of their classical role as lished from the three original families, but the familial binding partners of IGFs. For example, IGFBP1 (MIM occurrence prompted the designation of familial retinal 146730) is known to counteract apoptosis in hepatocytes artery macroaneurysms, or FRAM.11 Here, we expand the by binding to BAK, which impairs formation of the proa- clinical definition of this syndrome and show that it is poptotic p53/BAK complex,2 IGFBP3 (MIM 146732) upre- an autosomal-recessive condition caused by a founder gulates STAT1 to promote apoptosis in chondrocytes in mutation in IGFBP7 (MIM 602867). an IGF-independent manner,3 and IGFBP4 (MIM 146733) Patients with FRAM and their relatives were recruited directly binds to Fzd8 (MIM 606146) and LRP6 (MIM under an IRB-approved protocol (of the King Faisal Spe- 603507), making them less available to their ligand Wnt3 cialist Hospital and Research Center and the King Khaled and thus creating an inhibitory effect on the canonical Eye Specialist Hospital) with written informed consent. Wnt signaling.4 Patients had a thorough dysmorphologic, ophthalmologic, There is evidence to suggest a potential developmental and cardiologic evaluation. Eight Saudi families, for a total role for IGFBPs. IGFBP1, for example, has been shown to of 22 patients, were identified in the course of this study; regulate hypoxia-induced fetal growth retardation, IGFBP4 patient ages ranged from 4 to 37 years. The eight pedigrees is a cardiogenic factor, and Igfbp5 (MIM 146734) overex- were highly suggestive of an autosomal-recessive mode of pression confers increased neonatal mortality and retarded inheritance, and pseudodomiant inheritance was observed growth and muscle development.4–6 However, and in con- in one pedigree (Figure 1A). The classical presentation of trast to the demonstrated developmental role of the Igfs in macroaneurysms involving the retinal artery was seen in knockout mice, a similar role for any of the IGFBPs has not all patients, the youngest being 4 years of age, and some been easy to establish, in part because compensation and older patients have evidence of beading that extends redundancy may have masked a developmental pheno- well into the retinal periphery but at decreasing severity

1Department of Genetics, King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia; 2Vitreoretinal Division, King Khaled Eye Specialist Hospital, Riyadh 11462, Saudi Arabia; 3Department of Biostatistics, Epidemiology and Computing, King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia; 4Department of Cardiac Sciences, King Khalid University Hospital and College of Medicine, Riyadh 11462, Saudi Ara- bia; 5Department of Comparative Medicine, King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia; 6Department of Biochemistry and Molecular Genetics, American University of Beirut, Beirut 1107 2020, Lebanon; 7Department of Pediatrics, American University of Beirut, Beirut 1107 2020, Lebanon; 8Department of Anatomy and Cell Biology, College of Medicine, Alfaisal University, Riyadh 11533, Saudi Arabia; 9Department of Pediatrics, King Khalid University Hospital and College of Medicine, Riyadh 11462, Saudi Arabia 10These authors contributed equally to this work *Correspondence: [email protected] DOI 10.1016/j.ajhg.2011.07.010. Ó2011 by The American Society of Human Genetics. All rights reserved.

The American Journal of Human Genetics 89, 313–319, August 12, 2011 313 Figure 1. FRAM Is an Autosomal-Recessive Syndrome Defined by Retinal and Cardiac Phenotypes (A) Pedigrees of eight Saudi families with FRAM. Note the positive consanguinity in all families and the pseudodominant inheritance in family 004. (B) Representative fundus photo showing marked beading of the retinal artery on the optic nerve and along the major trunks. Many macroaneursyms are seen, either that are actively leaking with hard yellow exudate or that are cicatricial. (C) A short-axis view of the echocardiography, showing color turbulence with a Doppler pulse wave, giving a gradient of 20 mmHg at the supravalvular level of the pulmonary outflow tract. (D) Clinical photograph of a patient with FRAM; note the lack of facial dysmorphism.

(Figure 1B). In addition, a unique phenotype of supravalv- refined the region to a smaller locus (~4.7Mb) flanked by ular pulmonic stenosis was seen in all patients who under- rs76451666 and D4S398; this locus contained five anno- went echocardiography (nine declined examination) and tated genes and one hypothetical gene (Figure 2). Our anal- often required surgical correction due to its adverse hemo- ysis of the genes in the minimal linkage interval (n ¼ 6) only dynamic consequences (Figure 1C). No obvious dysmor- revealed the presence of previously reported SNPs (TT phic features or evidence of other system involvement rs115656299 and TT rs10866440 in REST [MIM 600571]; was noted (Figure 1D). Two patients died in the course of and AA rs1718878 and CC rs1718866 in POLR2B [MIM the study as a result of complications of their pulmonic 180661]) except in IGFBP7, which was found to harbor stenosis and suspected congestive hepatopathy. a splicing variant c.830-1G>A (NM_001553.1) (Figure 2). Whole-blood DNA was extracted, followed by genotyp- This consensus acceptor-site mutation segregated perfectly ing on Affy 250StyI SNP Chip per the manufacturer’s with the disease in all families. Microsatellite analysis and protocol (Affymetrix, Santa Clara, CA, USA). A homozy- analysis of additional SNPs obtained when the samples gosity scan was performed with Genotyping Console, and were run on the denser Axiom array (Affymetrix) confirmed linkage analysis was performed on easyLINKAGE via an the presence of a common haplotype that is bordered by autosomal-recessive model with 100% penetrance as previ- D4S1592 and rs12501656 and spans 96 SNPs over a ously described.12 Each of the eight families showed a run of 351kb genomic interval (Table S1). Screening of 300 Saudi homozygosity (ROH) on chromosome 4. Collectively, a controls (600 chromosomes) revealed the presence of LOD score of 11.4 was obtained for a locus delimited by only one heterozygous carrier, whose genotypic analysis SNPs rs6833480 and rs10017149 (~5.1 Mb) (Figure S1). confirmed that he harbors this mutation on the same Analysis with microsatellites and additional SNPs further disease haplotype, making him a likely carrier.

314 The American Journal of Human Genetics 89, 313–319, August 12, 2011 Figure 2. FRAM Is Caused by a Founder Splicing Mutation in IGFBP7 (A) Linkage analysis showing a very high LOD score of 11.4 on chromosome 4 on the first five families combined. Dotted lines emerging from the highest peak demarcate the critical linkage interval, which is further narrowed by the heterozygosity observed for flanking markers rs76451666 and D4S398, which only encompasses six genes, including IGFBP7 (shown in red with sequence chromatogram indicating the site of the G>A transition by a red asterisk). The common founder haplotype is flanked by D4S1592 and rs12501656. (B) RT-PCR and 30RACE, respectively, confirm the skipping of exon 5 and the lack of polyadenylation of the resulting aberrant transcript. (C) Immunoblot analysis shows more intense (130%) IGFBP7 in patient lymphoblasts (family F005 II-1, II-2 and II-4, from left to right) than in the control.

RT-PCR with a reverse primer on exon 5 confirmed that a lack of polyadenylation signal from exon 4, which re- this mutation fully abolishes the normal splice acceptor sulted in a lack of poly-A tail addition to the aberrant tran- site in that no product could be seen in patients (Figure 2). script that lacks exon 5. To circumvent this limitation, we In order to investigate the full extent of aberrant splicing, performed RT-PCR by using a forward primer in exon 1 and we performed a 30RACE experiment, which failed to a reverse primer in intron 4 (data not shown). The result recover any IGFBP7-specific transcript from the patient, indicated that in the patient, but not in the control, tran- whereas the expected transcript was readily identified in scription continues into intron 4 and introduces a stop the control (Figure 2). This is most likely explained by codon 7 bp downstream of the exon 4/intron 4 junction.

The American Journal of Human Genetics 89, 313–319, August 12, 2011 315 This aberrant transcript, therefore, predicts loss of the last five amino acids at the C terminus of an otherwise 282 aa IGFPB7 protein (29.13 kD) and their replacement by two novel amino acids for a total size of 279 aa (28.86 kD). At the protein level, immunoblot analysis with an antibody against the domains encoded by exons 3 and 4 of IGFBP7 consistently revealed a more intense band in patients than in controls (Figure 2). The reason for this increase is un- clear, but one possible explanation is defective secretion of the mutant protein. Another possibility is that the small truncation in the C terminus of the mutant protein re- moves a negative-feedback effect that is normally exerted so that the level of IGFBP7 is kept in the normal range. The above experimental evidence that no normal IGFBP7 transcript is made in blood cells from FRAM patients who are homozygous for this mutation makes it likely that the mutation we report is pathogenic. In addi- tion, this mutation is not observed in any of the public SNP databases, including that of the 1000 Genomes Project. Our finding of a single heterozygous individual out of 300 Saudi controls should not be viewed as evidence that this is a Saudi-specific benign variant because haplo- type analysis of this individual confirmed that he harbors the same disease haplotype in the heterozygous state, making him a likely carrier of a disease mutation. In order to examine IGFBP7 expression and its relevance to the phenotype in FRAM patients, we performed whole- mount in situ hybridization (WISH) experiments by using riboprobes for Igfbp7 on mouse embryos with InsituPro VSi (Intavis, Koeln, German) according the manufacturer’s protocol. This showed that Igfbp7 is indeed expressed in Figure 3. Igfbp7 Is Expressed in the Retinal Vasculature (A) WISH on E12 mouse embryos showing expression domains in the eye (both lens and retina) (Figure 3). Although these the eye (lens and surrounding retina); a sense probe is shown as results support a restricted pattern of expression, they do a negative control. not provide a robust link to the retinal phenotype seen (B) A flat-mount retina is used for immunohistochemistry with in FRAM patients because the murine retinal vasculature Igfbp7 antibody and isolectin-IB4 stain (latter is a marker for blood vessels). Note the positive staining in the superficial scans (1–3), develops postnatally. Indeed, longitudinal follow up of which include major smooth-muscle-invested retinal vasculature, the retinal phenotype in FRAM suggested that it is only but the largely negative staining in the deeper scans (4–6), where discernible at 4 years of age. Therefore, we resorted to im- smaller capillaries are more common. This is further confirmed in munohistochemistry analysis on retinal flat mounts and sections where specific Igfbp7 is shown surrounding larger vessels (7–9, inset). Staining with only secondary antibody shows no sections prepared from adult mice and with rat anti-mouse Igfbp7 staining (10–12). Igfbp7 antibody (R&D, catalog number MAB21201), which revealed an interesting expression pattern in the p value < 0.05, and we used Benjamini-Hochberg step-up retinal vasculature, where Igfbp7 is localized to the larger, procedure to cap the false-discovery rate at 5%).14 To verify smooth-muscle-invested vasculature but not in the smaller that the increased level of the components of the Ras/ capillaries. Localization surrounding the vessels is compat- MAPK pathway is associated with increased signaling, ible with extracellular secretion of Igfbp7 (Figure 3). which is characteristic of Noonan-spectrum disorders The overlapping cardiac features of FRAM (supravalvular (Rasopathies), we show that the active phosphorylated pulmonic stenosis) with that of Noonan spectrum disor- form of EPHB2 (most commonly known as ERK [MIM ders (mostly valvular pulmonic stenosis, but supravalvular 600997]) is markedly increased, whereas the level of total location has also been observed13) led us to investigate ERK remains unaltered (Figure 4; see also Figure S2). a potential mechanistic link. Indeed, global gene-expres- In 2002, Dhindsa and Abboud described an apparently sion profiling of patient lymphoblasts (performed in tripli- novel entity that they referred to as familial retinal artery cates with Affymetrix’s GeneChip Human Genome U133 macroaneurysm (FRAM) in three unrelated families (fami- Plus 2.0 Arrays) was consistent with upregulation of lies 003 and 004 in this study and one that was lost for BRAF and downstream targets of the Ras/MAPK pathway follow-up).11 The condition was most unusual in that (significantly modulated genes were defined as those aneurysms specifically of the retinal artery were seen at with an absolute n-fold change of n > 2 and adjusted a very young age. No conclusion was made about the

316 The American Journal of Human Genetics 89, 313–319, August 12, 2011 Figure 4. FRAM Is Characterized by Increased Signaling of the BRAF/MEK/ERK Pathway (A) IPA analysis led to the generation of a gene- interaction network of genes differentially ex- pressed in patients. Nodes represent genes, the shape of the node represents the functional class of the gene product, and the edges indicate the biological relationship between the nodes. Green indicates downregulation, and red indicates up- regulation in patients compared to controls. The color intensity is correlated with the n-fold change. (B) Upregulation of BRAF and p-ERK is confirmed at the protein level in patient lymphoblasts (family F005 II-1, II-2, and II-4, from left to right) compared to those of two controls. BRAF shows 100% increased intensity, and p-ERK shows 170% increased intensity in patients compared to two controls.

BRAF/MEK/ERK upregulation, which is also supported by immunoblot analysis. The observation that FRAM patients only lack a small part of the C terminus of IGFBP7 is intriguing because very little is known about the functional importance of the C terminus in IGFBPs other than that it contains an immunoglobulin-like domain.15 Thus, FRAM patients represent mode of inheritance or the possibility of systemic involve- a unique opportunity to observe the functional conse- ment, although magnetic resonance angiography (MRA) quences of the loss of that domain. did rule out intracranial vascular involvement. Here, we Before it was labeled as a gene encoding an insulin-like- improve the clinical delineation of this syndrome by high- growth-factor-binding protein, IGFBP7 was originally lighting the following two additional features: (1) It is described as MAC25 (meningioma-associated cDNA), a autosomal-recessive in origin, and the mother-to-child differentially expressed gene in meningioma and breast transmission in the original report was in fact a case of cancer tissues, and the protein was identified as tumor- pseudodominance, and (2) it is associated with supravalv- derived cell adhesion factor (TAF), also called angiomodu- ular pulmonic stenosis. lin.16,17 More recently, two major biological functions Despite the apparent lack of relatedness between the have emerged for this protein. The first is related to its eight study families and their different tribal and geo- tumor suppressor action, most notably in melanoma, graphic backgrounds, our genetic analysis strongly sug- where it was shown to mediate BRAFV600E-induced senes- gests that the disease mutation is of common ancestral cence by blocking the BRAF-MEK-ERK signaling.18–20 The origin, and the very small shared haplotype makes it likely second is related to its ability to block VEGF-induced that it is also ancient in origin. angiogenesis in a manner that is not mediated via promo- The mutational mechanism of c.830-1G>A in FRAM is tion of apoptosis but rather via the blocking of VEGF- not straightforward. One possibility is that the truncated induced activation of cyclooxygenase (COX)-2.21 How- IGFBP7 (which we show to be stably produced) acts in ever, despite these highly interesting biological actions of a dominant-negative manner. This is unlikely, however, IGFBP7, more comprehensive analysis has been limited because IGFBP7 is not known to dimerize and because by a lack of an animal model until recently, when the heterozygous carriers are disease free. Similarly, the possi- zebrafish ortholog was knocked down and the resulting bility that this is a neomorphic mutation is unlikely given morphants displayed a remarkable defect of vascular the lack of manifestations in any of the many carriers we sprouting and patterning.15 evaluated clinically in the course of this study. Loss-of- FRAM, therefore, represents a unique opportunity to function is the mechanism we prefer not only because it observe the consequences of germline mutations in is the usual mutational mechanism in autosomal-recessive IGFBP7 on vasculogenesis and angiogenesis. The highly diseases but also, and more importantly, because transcrip- penetrant phenotype of supravalvular pulmonic stenosis tomic analysis of lymphoblasts from FRAM patients is consistent with the vascular patterning defect seen in showed a pattern highly consistent with what is known zebrafish.15 The upregulation of BRAF/MEK/ERK signaling about IGFBP7 knockdown experiments, i.e., it showed provides a tantalizing pathway on which both FRAM

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