J Busch et al. SLCO2A1 Mutations Cause Primary Hypertrophic Osteoarthropathy

Mutations in the Prostaglandin Transporter SLCO2A1 Cause Primary Hypertrophic Osteoarthropathy with Digital Clubbing

Journal of Investigative Dermatology (2012) 132, 2473–2476; doi:10.1038/jid.2012.146; published online 14 June 2012

TO THE EDITOR noids (e.g., PGE2), this transport is in exon 9 (Figure 2). At age 25, he Digital clubbing with enlargement of mediated by the PG carrier SLCO2A1 complained about swelling of fingers the distal phalanges and overcurvature (Kanai et al., 1995). In a second step, and toes, thickening and furrowing of of the nails frequently occurs as a oxidation of prostanoids takes place facial skin, and increasing knee pain, secondary feature of chronic hypoxia within the cell via HPGD. In this study, which did not significantly improve in bronchopulmonary or cardiovascular we hypothesized that PHO can also be despite administration of nonsteroidal disease and systemic inflammatory or caused by mutations in the gene en- anti-inflammatory drugs. Seborrhea was neoplastic processes. Genetic causes coding the PG transporter SLCO2A1. successfully treated with 20 mg per day are much less common, but their SLCO2A1 is located on 3q22, com- isotretinoin. His renal PGE2 clearance decipherment provides a better under- prises 14 exons, and encodes a 643- was considerably increased (731 ng per standing of those more common dis- amino-acid type IV transmembrane hour per 1.73 m2, normal level 4–27), eases. Mutations in the 15-hydroxy- protein that belongs to the solute carrier similar to PGE-M, which showed a high prostaglandin dehydrogenase (HPGD) organic anion transporter family (Lu clearance of 2,960 ng per hour per gene encoding the major prostaglandin and Schuster, 1998). SLCO2A1 is 1.73 m2 (normal 62–482).

(PG) PGE2-catabolizing enzyme were broadly expressed in fetal and adult In another consanguineous Indian the first reported cause of primary tissues, and it contains 12 putative pedigree (Figure 1), we identified in both hypertrophic osteoarthropathy (PHO), transmembrane domains, three known affected brothers (26- and 28-year-old) whose most prominent feature is digital N-glycosylation sites, and one KAZAL- the homozygous missense mutation clubbing (Uppal et al., 2008; Tariq like domain. We performed direct c.763G4A (p.Gly255Arg) in exon 6. et al., 2009; Diggle et al., 2010; sequencing of the coding region and Digital clubbing with hyperhidrosis and Bergmann et al., 2011). Very recently, adjacent exon–intron boundaries of pain and swelling in ankle and knee Zhang et al. (2012) identified SLCO2A1 SLCO2A1 on an ABI 3500xL genetic joints started at about age 17 years. mutations in patients with autosomal analyzer (Applied Biosystems, Darm- In a 53-year-old Japanese patient, we recessive PHO. Intriguingly, individuals stadt, Germany) in seven PHO patients identified two heterozygous mutations: with PHO share many characteristics of five unrelated pedigrees who were c.1668G4C (p.Gln556His) in exon 12 with patients who display HO second- previously found to be negative for and c.940 þ 1G4A, which affects the ary to systemic pathologies. In addition mutations in HPGD. Primer sequences canonical donor splice site of intron 7. to digital clubbing, these are glan- and conditions are available on request. At the age of 20 years, he recognized dular hypertrophy and osteoblast proli- Blood samples were collected from all enlarged wrists and ankles, with digital feration, which cause thickening of patients and family members, and clubbing and arthralgia in both knees. the skin, bothersome palmoplantar genomic DNA was extracted using X-rays showed periostosis with cortical hyperhidrosis, and joint disease (pachy- standard procedures. Institutional ap- thickening and ectopic ossification. dermoperiostosis). Excessive sebum pro- proval for the experiments were ob- PGE2 levels were significantly elevated duction (seborrhea) may lead to severe tained, and written patient consent and (1,762 pg ml1 in serum, normal range facial acne. Acroosteolysis of the distal protocols were in accordance with the 25–200; and 7,063 ng mmol1 creatinine phalanges of the fingers and toes due to Declaration of Helsinki Principles. All in urine, normal level o50; Cayman, osteoclast stimulation is also commonly patients were of normal intelligence Cayman Biochemical, Ann Arbor, MI). observed (Castori et al., 2005). and height, and did not suffer from In a consanguineous Japanese pedi- Termination and clearance of PG pulmonary disease or any other inter- gree with two affected brothers, 21 and signaling is regulated by a two-step fering disease. 19 years of age, we identified the above process (Nomura et al., 2004). First, The 27-year-old male index patient of splicing mutation c.940 þ 1G4A in the extracellular PGs are taken up by a consanguineous family 1 from India homozygous state (Figure 2). PGE2 carrier-mediated process across the (Figure 1) carries the homozygous 1-bp levels measured with the same enzyme plasma membrane. For many prosta- deletion c.1292delC (p.Pro431LeufsX49) immunoassay kit were even more in- creased in these patients. Levels were Abbreviations: HPGD, 15-hydroxyprostaglandin dehydrogenase; NMD, nonsense-mediated decay; as follows (elder brother first): 2,375 1 PG, prostaglandin; PHO, hypertrophic osteoarthropathy and 17,701 pg ml in serum, and

www.jidonline.org 2473 J Busch et al. SLCO2A1 Mutations Cause Primary Hypertrophic Osteoarthropathy

Family 1 1°

ab

Family 2 3° Family 4 1°

a a

b b

Figure 1. Pedigrees and phenotypes of affected individuals with SLCO2A1 mutations. Top left: clinical features of the propositus of family 1. (a) Paw-like hands with moderate pachydermia, digital clubbing, and overcurvature of fingernails. (b) Clubbing of toes and moderate lymphoedema of lower legs. Top right: punch biopsy of the forehead. The epidermis shows normal differentiation, whereas the upper dermis demonstrates a dense diffuse inflammatory infiltrate composed of lymphocytes, plasma cells, and eosinophils. Some blood vessels show fibrinoid degeneration and neutrophils perivascularly with signs of cytoclasia, and some show extravasated erythrocytes. Lymph vessels appear dilated, and mild perifollicular fibrosis can be observed. Sebaceous glands are prominent and hair follicles contain demodex mites. Periodic acid Schiff (PAS) staining showed no other infectious agents. Mucin deposits are absent. Bottom left: (a) X-rays of ankle joints and hands of the elder affected brother of family 2 displaying extensive soft-tissue swelling with irregular periosteal proliferation, cortical thickening, and acroosteolysis. (b) Digital clubbing of the two affected brothers. Bottom right: (a, b) thickening and furrowing of the facial skin and digital clubbing of the hands of both affected brothers in pedigree 4 (a, elder brother, b, younger one). Subjects in the photographs have provided written, informed consent for the publication of their photographs.

9,260 and 17,701 ng mmol1 creatinine mutation represents an ancient founder ism), this mutation would result in an in urine, respectively. Other PG meta- allele rather than a recurrent mutation, out-of-frame transcript and probably bolites were not measured. as our patients harbor identical nonsense-mediated decay (NMD). The Japanese families, both carrying single-nucleotide polymorphisms, most NMD would be identical with loss-of- c.940 þ 1G4A, are not known to be probably in cis on the same allele function, i.e., a ‘‘most likely devastating related. Interestingly, this splicing muta- (rs10935090, rs4634113, rs6767412, chatacter’’ of this mutation. NMD can tion was also present in the heterozy- rs34550074, rs117837593, http://genome. also be postulated for c.1292delC gous state in one of the Han Chinese ucsc.edu). c.940 þ 1G4A destructs the (p.Pro431LeufsX49), identified homo- patients described recently (Zhang canonical donor splice site of intron 7. zygously in family 1. et al., 2012). Although we cannot prove If the splice resulted from exon skipping Overall, it might be hypothesized that this, it might be hypothesized that this (the most common splicing mechan- all or most SLCO2A1 mutations represent

2474 Journal of Investigative Dermatology (2012), Volume 132 J Busch et al. SLCO2A1 Mutations Cause Primary Hypertrophic Osteoarthropathy

a Extracellular 52 72 123 163 236 255 343 364 415 519 576 611 Plasma membrane

196995233 216 279 322 387 392 541 551 629 Intracellular

1 643

Family 1 Family 2 Family 3 Family 4 c.1292delC c.763G>A c.940+1G>A c.940+1G>A (p.Pro431LeufsX49) (p.Gly255Arg) Mut Mut Mut Mut

ACCCCC T AGG TA TGGATTAGAGCC T TTAAACRTAACTG TTAAACA TAACTG WT WT Mut WT

ACCCCCCTAGGTA TGGATTGGAGCCT GGTGCAST T CTTG TTAAACGTAACTG c.1668G>C (p.Gln556His) b p.G255R p.Q556H

Homo sapiens GDPRWIGAWWLGL SFAIGVQF LLMRL Canis lupus GDPRWIGAWWLGL SFAIGVQFLLMRL Bos taurus GDPRWIGAWWLGL SFAIGVQFLLMRL Mus musculus GDPRWIGAWWLGL SFAIGVQFLLMRL Rattus norvegicus GDPRWIGAWWLGL SFAIGVQFLLMRL Gallus gallus QDQRWIGAWWLGL SFAIGVQFLLMRL Caenorhabditis elegans HDEHWIGAWWLGF VIALSLAS FITNL

c d

p.Gly255Arg

p.Gln556His

Glutamine

Glycine Histidine

Arginine

Figure 2. Electropherograms and localization and effect of identified SLCO2A1 mutations in the prostaglandin transporter protein. Top: (a) scheme of the prostaglandin transporter SLCO2A1 (not drawn to scale). The SLCO2A1 protein contains 12 transmembrane domains with intracellularly located N and C terminus, 11 connecting loops in between, 3 glycosylation sites (highlighted as small circles), and a KAZAL-like domain (highlighted in bluish). The positions of the amino acids affected by mutations identified in this study are indicated by circles (in yellow and red for the two missense mutations G255R and Q556H), a green triangle (for the mutation c.940 þ 1G4A affecting the donor splice site of intron 7), and a blue square (for the frameshift mutation p.Pro431LeufsX). Below are depicted the respective electropherograms of all SLCO2A1 mutations identified in this study. The propositus of family 3 carried two heterozygous mutations, whereas the patients of the other families showed homozygosity for this locus in line with parental consanguinity in all three pedigrees. Middle: (b) conservation of the amino acids Gly (G) 255 and Gln (Q) 556 in SLCO2A1. The two missense mutations p.G255R and p.Q556H affect evolutionarily highly conserved amino acids in the SLCO2A1 protein (highlighted in yellow and red). Bottom left: (c) protein modeling of the missense mutation p.Gly255Arg in comparison with the wild-type (WT) sequence of SLCO2A1. Illustrated is the amino-acid region comprising amino acids 35–392 (only the protein ribbon is pictured). Both sequences (WT in green, mutated sequence in blue) put on top of each other. The corresponding position (Gly255) is marked in different colors (WT: yellow; mutated amino acid: red). The protein conformation has changed considerably; glycine (polar, neutral) present in the WT sequence is known to be much smaller than the basic amino acid arginine present in the mutant protein, whose side chain consists of a three-carbon aliphatic straight chain. Bottom right: (d) protein modeling of the missense mutation p.Gln556His in comparison with the WT sequence of SLCO2A1 according to the given illustration for p.Gly255Arg. Shown is the protein region comprising amino acids 529–579. WT glutamine is a polar and neutral amino acid that has been exchanged by the basic residue histidine with an imidazole ring, causing marked conformational changes.

www.jidonline.org 2475 EF Knol et al. Lymphocyte Function in Atopic Dermatitis

truncating alleles or affect residues that CONFLICT OF INTEREST Bordoli L, Kiefer F, Arnold K et al. (2009) Protein JB, VF, NB, HB, and CB are employees of structure homology modeling using SWISS- are indispensable for PG transporter MODEL workspace. Nat Protoc 4:1–13 activity, such as the missense muta- Bioscientia, a member of Sonic Healthcare. Castori M, Sinibaldi L, Mingarelli R et al. (2005) tions c.763G4A (p.Gly255Arg) and Pachydermoperiostosis: an update. Clin Gen- ACKNOWLEDGMENTS c.1668G4C (p.Gln556His) identified. et 68:477–86 We thank the families for their cooperation and Both changes are nonconservative, affect interest in the study. Diggle CP, Carr IM, Zitt E et al. (2010) Common evolutionarily highly conserved amino and recurrent HPGD mutations in Caucasian Jutta Busch1, Valeska Frank1, acids (Figure 2), and were predicted individuals with primary hypertrophic Nadine Bachmann1, Atoshi Otsuka2, osteoarthropathy. Rheumatology (Oxford) in silico by all bioinformatic tools of Vinzenz Oji3, Dieter Metze4, 49:1056–62 pathogenic relevance (Supplementary Ta- Krati Shah5, Sumita Danda5, Kanai N, Lu R, Satriano JA et al. (1995) ble 1 online). The pathogenic character Identification and characterization of a pros- Bernhard Watzer6, Heiko Traupe3, of c.763G4A (p.Gly255Arg) and the taglandin transporter. Science 268:866–9 Hanno J. Bolz1,7, Kenji Kabashima2 and impact of the glycine residue at position Lu R, Schuster VL (1998) Molecular cloning of the Carsten Bergmann1,8,9 255 is further strengthened by the fact gene for the human prostaglandin transporter 1 hPGT: gene organization, promoter activity, that the same codon was mutated Center for Human Genetics, Bioscientia, Ingelheim, Germany; 2Department of and chromosomal localization. Biochem (c.764G4A; p.Gly255Glu) in a patient Dermatology, Kyoto University, Kyoto, Japan; Biophys Res Commun 246:805–12 described by Zhang et al. (2012). To 3Department of Dermatology, University Meng EC, Pettersen EF, Couch GS et al. (2006) corroborate these data, we constructed Hospital Mu¨nster, Mu¨nster, Germany; Tools for integrated sequence-structure ana- 4 lysis with UCSF Chimera. BMC Bioinform molecular models of wild-type and Department of Pathology, University Hospital Mu¨nster, Mu¨nster, Germany; 5Clinical 7:339 mutated SLCO2A1 proteins to visualize Genetics Unit, Christian Medical College, Nomura T, Lu R, Pucci ML et al. (2004) The two- changes of protein folding and structure Vellore, India; 6Department of Pediatrics, step model of prostaglandin signal termina- (Meng et al., 2006; Bordoli et al., 2009; University Marburg, Marburg, Germany; tion: in vitro reconstitution with the prosta- Yang et al., 2011). Both mutations are 7Department of Human Genetics, University of glandin transporter and prostaglandin 15 8 dehydrogenase. Mol Pharmacol 65:973–8 located in a transmembrane domain Cologne, Cologne, Germany; Department of Human Genetics, RWTH Aachen University, Tariq M, Azeem Z, Ali G et al. (2009) Mutation in and considerably affect the protein Aachen, Germany; 9Center for Clinical the HPGD gene encoding NAD+ dependent structure (Figure 2), which was addi- Research, University Hospital Freiburg, 15-hydroxyprostaglandin dehydrogenase un- tionally confirmed by Ramachandran Freiburg, Germany derlies isolated congenital nail clubbing plots (Supplementary Figure S1 online). E-mail: [email protected] (ICNC). J Med Genet 46:14–20 In this report, we show that Uppal S, Diggle CP, Carr IM et al. (2008) Mutations in 15-hydroxyprostaglandin dehy- SLCO2A1 is a frequent cause of auto- SUPPLEMENTARY MATERIAL drogenase cause primary hypertrophic os- somal recessive PHO, confirming the Supplementary material is linked to the online teoarthropathy. Nat Genet 40:789–93 data recently published by Zhang et al., version of the paper at http://www.nature.com/jid Yang Z, Lasker K, Schneidman-Duhovny D et al. 2012. Although most patients harbor (2011) UCSF Chimera, MODELLER, and IMP: mutations in HPGD and SLCO2A1, REFERENCES an integrated modeling system. J Struct Biol; e-pub ahead of print 22 September 2011 further genetic heterogeneity is likely, Bergmann C, Wobser M, Morbach H et al. (2011) and candidate approaches targeting Primary hypertrophic osteoarthropathy with Zhang Z, Xia W, He J et al. (2012) Exome digital clubbing and palmoplantar hyperhi- sequencing identifies SLCO2A1 mutations other PG-signaling components seem drosis caused by 15-PGHD/HPGD loss-of- as a cause of primary hypertrophic osteoar- promising. function mutations. Exp Dermatol 20:531–3 thropathy. Am J Hum Genet 90:125–32

Modulation of Lymphocyte Function In Vivo via Inhibition of Calcineurin or Purine Synthesis in Patients with Atopic Dermatitis

Journal of Investigative Dermatology (2012) 132, 2476–2479; doi:10.1038/jid.2012.150; published online 24 May 2012

TO THE EDITOR the immune response, production of In AD, activated T cells can be found In atopic dermatitis (AD), lymphocytes Igs, and sustaining cutaneous inflam- in blood, and most importantly in have an important role via regulation of mation. affected skin a marked increase of activated T cells is present (Akdis et al., 2006). Serum levels of T-cell Abbreviations: AD, atopic dermatitis; CsA, cyclosporine A; EC-MPS, enteric-coated mycophenolate sodium; FUCT-VII, fucosyl transferase VII; MPA, mycophenolic acid; TARC, thymus and activation chemokines CCL17 (thymus and activa- regulated chemokine; TNF–AIP3, tumor necrosis factor–alpha inducible protein-3 tion regulated chemokine, TARC) and

2476 Journal of Investigative Dermatology (2012), Volume 132