European Journal of Human Genetics (2009) 17, 1570 – 1576 & 2009 Macmillan Publishers Limited All rights reserved 1018-4813/09 $32.00 www.nature.com/ejhg ARTICLE HPGD mutations cause cranioosteoarthropathy but not autosomal dominant digital clubbing Wenke Seifert1,2,3,10, Julia Beninde4,10, Katrin Hoffmann1, Tom H Lindner5, Christian Bassir6, Fuat Aksu7, Christoph Hu¨bner8, Nienke E Verbeek9, Stefan Mundlos1 and Denise Horn*,1 1Institute of Medical Genetics, Charite´, University Medicine of Berlin, Berlin, Germany; 2Cologne Center for Genomics, Universita¨t zu Ko¨ln, Ko¨ln, Germany; 3Department of Biology, Chemistry, and Pharmacy, Free University of Berlin, Berlin, Germany; 4Center of Developmental Neurology and Epileptology, Frankfurt, Germany; 5Division of Nephrology, Department of Internal Medicine, University Clinic Leipzig, Leipzig, Germany; 6Department of Pediatric Radiology, Charite´, University Medicine of Berlin, Berlin, Germany; 7Department of Neuropediatrics, University of Witten/ Herdecke, Datteln, Germany; 8Department of Pediatric Neurology, Charite´, University Medicine of Berlin, Berlin, Germany; 9Department of Medical Genetics, Universitary Medical Center Utrecht, Utrecht, The Netherlands Cranio-osteoarthropathy, clinically classified as a variant of primary hypertrophic osteoarthropathy, is a very rare autosomal-recessive condition characterized by delayed closure of the cranial sutures and fontanels, digital clubbing, arthropathy, and periostosis. Recently, mutations in the gene HPGD, which encodes the NAD þ -dependent 15-hydroxyprostaglandin dehydrogenase, were reported in four families affected with primary hypertrophic osteoarthropathy and one family with autosomal-recessive isolated nail clubbing. We report the clinical and molecular findings in four patients from two families affected with cranio-osteoarthropathy and one family with isolated, autosomal dominant digital clubbing. Genome- wide homozygosity mapping identified a locus for cranio-osteoarthropathy harboring the HPGD gene in one affected family. We detected two novel homozygous mutations in HPGD in these families: a missense mutation affecting the NAD þ binding motif and a frameshift mutation. The clinical presentation in our patients was variable. Digital clubbing and hyperhidrosis were present in all cases. Delayed closure of the cranial sutures and fontanels, periostosis, and arthropathy were not consistent clinical features. No HPGD mutation was detected in a familial case of autosomal dominant isolated digital clubbing. The failure to identify any mutation in a family with an autosomal dominant type of isolated digital clubbing suggests that HPGD is not the major gene for this condition. European Journal of Human Genetics (2009) 17, 1570–1576; doi:10.1038/ejhg.2009.104; published online 1 July 2009 Keywords: cranio-osteoarthropathy; pachydermoperiostosis; primary hypertrophic osteoarthropathy; HPGD gene Introduction neous and is characterized by digital clubbing, arthropathy, Primary hypertrophic osteoarthropathy (PHO, MIM and periostosis, which consists of new bone formation 259100, 167100) is genetically and clinically heteroge- around the periosteum of long bones. For PHO, both autosomal-recessive and dominant inheritance have *Correspondence: Dr D Horn, Institute of Medical Genetics, Charite´- 1 Universita¨tsmedizin Berlin, Augustenburgerplatz 1, Berlin, 13353, Germany. previously been postulated. In contrast, hyper- Tel: þ 49 30 4505 69132; Fax: þ 49 30 4505 69914; trophic osteoarthropathy (HO) develops as a secondary E-mail: [email protected] consequence to different acquired diseases such as 10These authors contributed equally to this work Received 25 February 2009; revised 12 May 2009; accepted 14 May 2009; intrathoracic tumors and shows clinical overlap with published online 1 July 2009 PHO. HPGD mutations cause cranioosteoarthropathy W Seifert et al 1571 Two variants of PHO have been distinguished clinically Material and methods (Supplementary Table 1). In addition to features of PHO, Patients pachydermoperiostosis (MIM 167100, 259100) includes Written informed consent was obtained from all partici- clinical signs of pachydermia, such as coarsening of the pants or their legal guardians. facial features as early as in adolescence, seborrheic 2 hyperplasia, skin thickening, and excessive sweating. Genome-wide linkage scan Cranio-osteoarthropathy (COA, Currarino idiopathic Genotyping was performed using the 250k Affymetrix SNP osteoarthropathy, MIM 259100) shows, in addition to the chip (GeneChip Mapping 250K Nsp Assay Kit, GeneChip other signs of PHO, poor neurocranium ossification with Mapping 250K Sty Assay Kit Affymetrix, Inc. (US), delayed closure of the cranial sutures and fontanels and Santa Clara, CA, USA) in family, 1. We analyzed the data 3–5 increased number of wormian bones. Clinical signs of using ALLEGRO v1.2c (http://www.decode.com/software/ 3 pachydermia are absent in patients with COA. An allegro/), GENEHUNTER v2.1r5 (http://www.broad.mit. autosomal-recessive mode of inheritance has been sug- edu/ftp/distribution/software/genehunter/) under the gested in COA by analysis of the affected pedigrees. graphical user-interface easyLINKAGE v5.08 (http://compbio. Emphasizing the clinical overlap between pachydermo- charite.de/genetik/hoffmann/easyLINKAGE/).10,11 We assumed periostosis and COA, O’Connell et al suggested that these a recessive model with complete penetrance, disease allele 6 disorders may be allelic. frequency of 0.001, and equally distributed marker alleles. Recently, homozygous mutations in the 15-hydroxy- To save analysis time and to avoid linkage disequilibrium- prostaglandin dehydrogenase (HPGD) gene located at related artifacts, we analyzed informative single nucleotide 7 4q33-q34 have been described to cause PHO. HPGD polymorphisms at every 0.5 cM along the genome. þ encodes the NAD -dependent 15-hydroxyprostaglandin We included all available family members of family 1 dehydrogenase (15-PGDH, EC 1.1.1.141), which is a and reconstructed haplotypes by ALLEGRO and prostaglandin E2 (PGE2) catabolizing enzyme. Mutations manually. Next, we sequenced functional candidate genes reported so far in PHO very likely result in loss of within the linkage interval by standard sequencing 12 enzymatic function. Elevated urine PGE2 levels were found procedures. in all homozygous and some of the heterozygous muta- tional carriers.7 These findings support the idea that Mutational analysis impaired metabolism of PGE is critical in PHO as well as 2 Genomic DNA was isolated from the peripheral blood in HO pathogenesis. PGE is a ubiquitous lipid mediator 2 using standard techniques. For mutation screening, we regulated by a number of enzymes, including different amplified the coding region of HPGD (NM_000860), COX enzymes. The COX enzymes are the pharmacological including the flanking intronic sequences and the pre- targets of non-steroidal anti-inflammatory drugs (NSAIDs). dicted promoter region. Primer sequences and PCR condi- Owing to this pathway, NSAIDs are able to block PGE 2 tions are available on request. PCR products were purified synthesis. Further studies should clarify whether progres- using the enzymes exonuclease I and shrimp alkaline sion of PHO could be influenced by NSAID treatment. phosphatase treatment, and directly sequenced with the HpgdÀ/À mice are affected by patent ductus arteriosus BigDye Terminator v3.1 Cycle Sequencing Kit (Applied and die shortly after birth because of high-output heart Biosystems, Darmstadt, Germany) and analyzed on failure, but do not show other manifestations of PHO.8 an automated DNA Analyzer (3730 Applied Biosystems). These findings in mice indicate that PGE2 metabolism by 15-PGDH is required for the remodeling of the ductus arteriosus. In humans, an increased frequency of 25% for a Molecular modeling of the 15-PGDH patent ductus arteriosus has been documented in patients The multiple sequence alignment was performed using with PHO, whereas the general risk is about 0.05 % in CLUSTALW implemented in the BioEDIT V7.0.5.3 (Tom full-term neonates.6,7 Hall, Ibis Biosciences, Carlsbad, CA, USA) program. For Recently, a homozygous missense mutation in HPGD studying the missense mutation G18C, we calculated its has been identified in a single family affected with putative effect on 15-PGDH (2GDZ.pdb) using the mutate isolated congenital nail clubbing as an autosomal-recessive tool followed by energy minimization and computation of trait.9 H-bonds, both implemented in the DeepView software In this study, we found significant linkage of COA to (Swiss Pdp Viewer). a region harboring the HPGD gene in one affected family and report novel mutations in HPGD causing COA in two families. However, in a third family with dominant Results inheritance of isolated digital nail clubbing, we found Clinical reports no mutation in HPGD indicating another underlying Family 1 This Turkish family is multiply consanguineous gene defect. and the affected patients were offspring of first-cousin European Journal of Human Genetics HPGD mutations cause cranioosteoarthropathy W Seifert et al 1572 parents (Supplementary Figure 1b). The affected propositus A female cousin of the mother (patient IV-1) who had (patient V-1) was first seen at the age of 13 months for mild first-cousin parents was also affected. During the first year developmental delay and skeletal anomalies, such as of life, she suffered from an atopic eczema that disappeared widening of the cranial sutures and clubbing of the distal