Mutations in CCBE1 Cause Generalized Lymph Vessel Dysplasia

brief communications

Mutations in CCBE1 cause (Fig. 1a–f)8. Subsequently, subjects with lymphangiectasias in pleura, pericardium, thyroid gland and kidney and with hydrops fetalis were generalized lymph vessel dysplasia described9,10. The entity was designated lymphedema-lymphangiectasia– mental retardation or Hennekam syndrome (MIM 235510). Occurrence in humans of affected siblings, equal occurrence among sexes and frequent consanguinity indicated autosomal recessive inheritance9. 1 2 2 1 Marielle Alders , Benjamin M Hogan , Evisa Gjini , Faranak Salehi , We collected blood samples from a series of 27 subjects with Hennekam 3 4 5 Lihadh Al-Gazali , Eric A Hennekam , Eva E Holmberg , syndrome born to 22 families. None of the subjects carried mutations 1 6 7,8 Marcel M A M Mannens , Margot F Mulder , G Johan A Offerhaus , in FLT4, FOXC2 or SOX18. We performed homozygosity mapping in 5 9 10 Trine E Prescott , Eelco J Schroor , Joke B G M Verheij , three unpublished subjects (A, B and C) originating from a small isolate 2 11,14 12 Merlijn Witte , Petra J Zwijnenburg , Mikka Vikkula , in The Netherlands. Pedigree analysis had shown the parents of subject 2,15 13–15 Stefan Schulte-Merker & Raoul C Hennekam A to be consanguineous and all three subjects to be related (Fig. 1g). We reasoned that occurrence of three cases of a rare disorder in a small Lymphedema, lymphangiectasias, mental retardation and isolate suggested homozygosity for a founder mutation. Homozygosity unusual facial characteristics define the autosomal recessive mapping identified a 5.7-Mb homozygous region on chromosome 18q21 Hennekam syndrome. Homozygosity mapping identified a with identical haplotypes in the three affected individuals (Fig. 1h and critical chromosomal region containing CCBE1, the human Supplementary Table 1). The region contained 29 genes. Additional ortholog of a gene essential for lymphangiogenesis in zebrafish. homozygosity mapping in two subjects (D, E) born to different consan Homozygous and compound heterozygous mutations in guineous parents11 (Fig. 1g) identified several stretches of homozygosity, seven subjects paired with functional analysis in a zebrafish including a segment on 18q21. The overlapping homozygous region was model identify CCBE1 as one of few genes causing primary 0.5 Mb long and contained four genes (Fig. 1h). generalized lymph-vessel dysplasia in humans. Of particular interest was CCBE1, encoding Collagen and Calcium- Binding EGF-domain-1, a secreted protein (Supplementary Figs. 1 The lymphatic system comprises a vascular system separate from the and 2) required for embryonic lymphangiogenesis in zebrafish12. cardiovascular system, essential for immune responses, fluid homeo Sequencing CCBE1 revealed homozygous mutations in all five subjects stasis and fat absorption. Lymphatic vessels develop in a complex pro (Supplementary Fig. 3 and Supplementary Table 2). Mutations in sub cess termed lymphangiogenesis that involves budding, migration and jects A, B and C (C75S) and subject D (C102S) are N-terminal of the proliferation of lymphatic endothelial progenitor cells1–3. A few genes, putative calcium-binding EGF domain. This region also shows EGF-like © All rights reserved. 2009 Inc. Nature America, such as FLT4 (ref. 4), FOXC2 (ref. 5) and SOX18 (ref. 6), are known to sequences containing cysteine residues that are highly conserved, suggest be critically involved in lymph vessel formation in humans. ing functional relevance. The mutation G327R in subject E is predicted to Disturbances of lymphangiogenesis usually cause disruption of the disrupt the glycine backbone in the putative collagen helix of CCBE1. drainage of interstitial fluids into the cardiovascular system, resulting Subsequently, we screened 19 more families with Hennekam syn in lymphedema, chylothorax or pleural effusion, chylous ascites, and drome for CCBE1 mutations and found compound heterozygous angiectasias of lymph vessels in intestines and other organs7. Signs of CCBE1 mutations in two affected individuals (F, G) (Supplementary lymph-vessel dysplasias are commonly limited to the limbs1. In 1989, an Fig. 3 and Supplementary Table 2): subject F carried a maternally inbred family was described in which four mentally retarded members inherited, single-nucleotide insertion, c.683_684insT, which intro had a widespread congenital lymphatic malformation syndrome with duces an in-frame premature stop codon, leading to either production limb lymphedema, and lymphangiectasias of the intestine and at a later of protein lacking the collagen domain or nonsense-mediated mRNA age also of the lungs8. In addition, affected individuals had unusual facial decay13. In addition, subject F carried a paternally inherited mis characteristics (flat face, flat and broad nasal bridge, hypertelorism) sense mutation, R158C. Subject G was compound heterozygous for thought to reflect the extent of early intrauterine facial lymphedema c.683_684insT and C174R (Supplementary Table 2). Both R158C and

1Department of Clinical Genetics, Academic Medical Centre, Amsterdam, The Netherlands. 2Hubrecht Institute – Koninklijke Nederlandse Akademie van Wetenschappen and University Medical Centre, Utrecht, The Netherlands. 3Department of Paediatrics and Pathology, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates. 4Department of Clinical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands. 5Department of Medical Genetics, Oslo University Hospital, Rikshospitalet, Oslo, Norway. 6Department of Pediatrics, Free University Medical Center, Amsterdam, The Netherlands. 7Department of Pathology, University Medical Centre Utrecht, Utrecht, The Netherlands. 8Department of Pathology, Academic Medical Center, Amsterdam, The Netherlands. 9Department of Pediatrics Amalia, Isala Clinics, Zwolle, The Netherlands. 10Department of Clinical Genetics, University Medical Centre Groningen, Groningen, The Netherlands. 11Department of Clinical Genetics, Free University Medical Center, Amsterdam, The Netherlands. 12Laboratory of Human Molecular Genetics, de Duve Institute, Université Catholique de Louvain, Brussels, Belgium. 13Clinical and Molecular Genetics Unit, Institute of Child Health, Great Ormond Street Hospital for Children, University College London, London, UK. 14Department of Pediatrics, Academic Medical Centre, Amsterdam, The Netherlands. 15These authors contributed equally to this work. Correspondence should be addressed to R.C.H. ([email protected]).

Received 8 June; accepted 13 October; published online 22 November 2009; doi:10.1038/ng.484

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a b c d

e f

g h Subject Mb AB C DE rs1434511

45 18q21.1

rs2928927

50 rs1145315 18q21.2

rs1506330 18q21.33 rs1381548 Subject E Subject D 60 rs1472948

Figure 1 Phenotypes of subjects with generalized lymphatic dysplasia and homozygosity mapping in five subjects with consanguineous parents. (a) Subject D, with flat face, hypertelorism and flat nasal bridge. (b) Subject G, with widespread congenital lymphedema and distended abdomen due to ascites. (c,d) Subject F, showing webbing of the neck as result of intrauterine lymphedema, limb lymphedema, distended abdomen due to ascites, and muscle wasting at 20 years. (e) Distended lymph vessels in the intestinal wall of subject F (see also Supplementary Fig. 6). (f) Uneven endothelial podoplanin staining in lymphangiectasia (small intestine, subject F). (g) Pedigrees of the subjects with consanguineous parents. Affected subjects are shown as filled circles (females) or squares (males). All parents tested were heterozygous carriers (dot). (h) Homozygous regions at chromosome 18q21, indicating a shared homozygous region of 0.5 Mb. Bars, homozygous segments; flanking SNPs are indicated.

C174R are in the calcium-binding EGF domain, the first introducing knockdown (morphant) phenotypes, whereas mutant mRNA does an extra cysteine that might interfere with proper folding of the pro not. Therefore this model is suitable for testing pathogenicity of tein and the second disrupting a conserved cysteine residue predicted missense mutations identified in this study. to form disulfide bonds important for the secondary structure of this We introduced homologous mutations in zebrafish ccbe1 and domain (Fig. 2a). All mutations were absent in controls of Western tested the ability to rescue the ccbe1 morphant phenotype (Fig. 2b–i). European (n = 100) or Arabic (to match the ethnic background of Injection of wild-type ccbe1 mRNA reliably rescued the absence of the subject D; n = 97) descent. None of the other subjects from 17 families thoracic duct (Fig. 2b–d,j). Three of the five mutant mRNAs tested carrying Hennekam syndrome harbored a CCBE1 mutation. (C94S, C166R and G313R, equivalent to human C102S, C174R and The function of ccbe1 (ref. 12) has been studied in a zebrafish lymph G327R, respectively) were not able to confer any rescue (Fig. 2f,h–j; angiogenesis model14,15. Zebrafish ccbe1 mutants lack parachordal Supplementary Fig. 4). Mutant C67S (equivalent to human C75S) lymphangioblasts and all known lymphatic vessels. Mutants develop showed weak rescue, and R150C (equivalent to human R158C) more lymphedema but retain a largely normal cardiovascular system. Wild- robust rescue comparable to that seen with wild type (Fig. 2e,g,j), indi type ccbe1 mRNA rescues the mutant12 and morpholino-induced cating that at least some mutant protein function was preserved. This

Nature Genetics volume 41 | number 12 | december 2009 1273 B rief communications

a 1 134 174 245 290 300 333 406 b Control c ccbe1 ATG MO d ccbe1 ATG MO Ca-EGF domain Collagen-1 Collagen-2 ccbe1 mRNA **

C75S C102SR158C C174RL229fsX8 G327R GAPGPRGSPGP Human KKCCKGY CEQQCTDNF LGSYRCECR DGKTCTRGD GAPGPRGSPGP ccbe1 ATG MO ccbe1 ATG MO ccbe1 ATG MO Chimp KKCCKGY CEQQCTDNF LGSYRCECR DGKTCTRGD e f g Mouse KKCCKGY CEQQCTDNF MGSYHCECR DGRTCTRGD GAPGPPGSPGP ccbe1 C675 mRNA ccbe1 C945 mRNA ccbe1 R150C mRNA Dog KKCCKGY CEQQCTDNF VGSYRCECR DGRTCTRGD GAPGPRGSPGP * Rat KKCCKGY CEQQCTDNF MGSYHCECR DGRTCTRGD GAPGPPGSPGP * * Cow KKCCKGY CEQQCTDNF VGSYRCECR DGRTCTKED GAPGPRGSPGP Chicken KKCCKGY CEQQCTDNF VGSYRCECH DGRTCTKGD GAPGPKGIPGP Zebrafish KKCCEGF CEQQCTDHF PGSYRCDCH DGKTCTKGE GFPGPSGPPGP **** ********* **** * *** ** **** **** ccbe1 ATG MO j 100% TD – h ccbe1 C166R mRNA 80% TD + Figure 2 Mutations in CCBE1 abolish normal gene function. (a) Locations * * 60% of mutations in conserved amino acids in CCBE1. Information on domain 40% localization was obtained from UniProt (http://www.uniprot.org). 20% (b–j) Functional analysis of the mutations in the zebrafish ccbe1 model ccbe1 ATG MO i 0% using the transgenic line TG(fli1a:gfp) y1, TG(kdr-l:ras-cherry)s916. Fli1a ccbe1 G313R mRNA MO (green) is a marker for both blood and lymphatic endothelial cells, * * whereas Kdr-l (red) is expressed only in blood vessels12. (b,c) Injection Uninjectedccbe1 of ccbe1 ATG-targeting morpholino (MO) (5 ng per embryo) led to a MO + wt mRNA MO + C67SMO + mRNAC945 mRNA robust phenocopy of the ccbe1 mutant phenotype with absence (*) of MO + R150CMO + C166RMO mRNA + G313R mRNA mRNA the thoracic duct in injected (c) but not uninjected control (b) embryos 5 d postfertilization. (d–i) The ccbe1 ATG MO phenotype is robustly rescued by injection of wild-type ccbe1 mRNA (d) but not mRNA encoding the C67S (e), C94S (f), C166R (h) or G313R (i) substitutions. The mRNA encoding the R150C substitution was capable of rescue (g). Asterisk, lack of rescue; arrows, thoracic duct. mRNAs were all injected at 350 pg per embryo. (j) Summary of rescue experiments. Embryos were scored for the presence or absence of the thoracic duct (TD), and data are shown for uninjected control (100% TD+, n = 21 embryos scored), MO injected (6% TD+, n = 75), MO + ccbe1 mRNA (97% TD+, n = 117), MO + ccbe1 C67S mRNA (25% TD+, n = 108), MO + ccbe1 C94S mRNA (4% TD+, n = 96), MO + ccbe1 R150C mRNA (78% TD+, n = 57), MO + ccbe1 C166R mRNA (0% TD+, n = 87) and MO + ccbe1 G313R mRNA (0% TD+, n = 73). The mutations in e–i are equivalent to human C75S, C102S, R158C, C174R and G327R, respectively.

does not exclude pathogenicity, as even if the mutant zebrafish protein Centre Amsterdam) provided gut biopsies of controls and H. Begthel (Hubrecht has reduced activity, administration in excess by high-dose mRNA Institute, Utrecht) performed histology. T. Nilsson (McGill University) generously GFP injection may still provide sufficient function to achieve rescue. provided the GalNAc-T2 cell line and A. Akhmanova (Erasmus Medical Center, Rotterdam) the NPY:mVenus cell line. We are grateful to the subjects and their Mutations in CCBE1 are described here in a subset (5/22 = 23%) families for their cooperation. of families carrying Hennekam syndrome, indicating genetic heterogeneity. CCBE1 is not expressed in endothelial cells of lymph AUTHOR CONTRIBUTIONS vessels 12, and it may be a component of the extracellular matrix. L.A.-G., E.E.H., M.F.M., T.E.P., E.J.S., J.B.G.M.V., P.J.Z. and R.C.H. analyzed affected individuals, and E.A.H. performed genealogical studies. M.A., F.S., In zebrafish, ccbe1 expression was observed along the earliest migra M.M.A.M.M., M.V., B.M.H., E.G., G.J.A.O., M.W. and S.S.-M. carried out tion routes of endothelial cells that sprout from the posterior cardinal experiments and analyzed data. S.S.-M. supervised the zebrafish experiments, vein and migrate circuitously before developing into lymphatic and R.C.H. supervised the clinical and molecular studies and initiated the study. vessels12,14. CCBE1 might therefore be involved in guidance of these M.A., S.S.-M. and R.C.H. wrote the manuscript. 12 migrating cells . Ccbe1 is expressed in the developing mouse intestine COMPETING INTERESTS STATEMENT © All rights reserved. 2009 Inc. Nature America, (Supplementary Fig. 5), and we identified very few lymphatic vessels The authors declare competing financial interests: details accompany the full-text in intestinal biopsies from human subjects (Supplementary Fig. 6), HTML version of the paper at http://www.nature.com/naturegenetics/. consistent with a requirement for CCBE1 in lymphangiogenesis. Mutations in as-yet-unknown interaction partners of CCBE1 may Published online at http://www.nature.com/naturegenetics/. cause a similar phenotype and are further candidate genes for this Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/. form of generalized lymphatic dysplasia in humans. All subjects or their legal representatives provided informed consent to the study. The study was approved by the medical ethical committee of the Academic Medical Centre in Amsterdam. Written 1. Karpanen, T. & Alitalo, K. Annual Rev. Pathol. Dis. 3, 367–397 (2008). 2. Oliver, G. & Alitalo, K. Annu. Rev. Cell Dev. Biol. 21, 457–483 (2005). permission was obtained from the subjects or their legal represen 3. Cueni, L.N. & Detmar, M. J. Invest. Dermatol. 126, 2167–2177 (2006). tatives to publish clinical pictures. 4. Ferrell, R.E. et al. Hum. Mol. Genet. 7, 2073–2078 (1998). 5. Fang, J. et al. Am. J. Hum. Genet. 67, 1382–1388 (2000). 6. Irrthum, A. et al. Am. J. Hum. Genet. 72, 1470–1478 (2003). Accession codes. GenBank: Homo sapiens CCBE1, NM_133459.1; 7. Hilliard, R.I., Mckendry, J.B.J. & Phillips, M.J. Pediatrics 86, 988–994 (1990). Danio rerio ccbe1, NM_001163923. 8. Hennekam, R.C. et al. Am. J. Med. Genet. 34, 593–600 (1989). 9. Van Balkom, I.D. et al. Am. J. Med. Genet. 112, 412–421 (2002). 10. Bellini, C. et al. Am. J. Med. Genet. 120A, 92–96 (2003). Note: Supplementary information is available on the Nature Genetics website. 11. Al-Gazali, L.I., Hertecant, J., Ahmed, R., Khan, N.A. & Padmanabhan, R. Clin. Dysmorphol. 12, 227–232 (2003). Acknowledgments 12. Hogan, B.M. et al. Nat. Genet. 41, 396–398 (2009). S.S.-M. and E.G. were supported by the Koninklijke Nederlandse Akademie 13. Maquat, L.E. Rev. Mol. Cell. Biol. 5, 89–99 (2004). van Wetenschappen, B.M.H. by an Australian National Health and Medical 14. Yaniv, K. et al. Nat. Med. 12, 711–716 (2006). Research Council CJ Martin Fellowship. M.J. Van De Vijver (Academic Medical 15. Küchler, A.M. et al. Curr. Biol. 16, 1244–1248 (2006).

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