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 mlÀ1 in serum, normal range facial acne. Acroosteolysis of the distal protocols were in accordance with the 25–200; and 7,063 ng mmolÀ1 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 À1 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 mmolÀ1 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),