A novel homozygous splice site mutation in the HPGD causes mild primary hypertrophic osteoarthropathy L. Sinibaldi1, G. Harifi2, I. Bottillo1,3, M. Iannicelli1,3, S. El Hassani2, F. Brancati1,4, B. Dallapiccola1,5

1CSS-Mendel Institute, Casa Sollievo della Sofferenza Hospital, Rome, Italy; 2Service de Rhumatologie, Centre Hospitalo-Universitaire Mohamed VI, Marrakech, Morocco; 3Department of Experimental , Sapienza University Rome, Rome, Italy; 4CeSI Aging Research Centre, Department of Biomedical Sciences, Gabriele d’Annunzio University, Chieti, Italy; 5Bambino Gesù Children’s Hospital. IRCCS, Rome, Italy.

Abstract Objectives Homozygous mutations in HPGD gene, encoding 15-hydroxyprostaglandin dehydrogenase, have recently been associated with primary hypertrophic osteoarthropathy (PHO). So far, only 7 HPGD alterations are known. In order to expand this mutational spectrum and better delineate the HPGD-related phenotype, we report the clinical and molecular characterisation of a 13-year-old boy and compare his features to known mutated patients.

Methods The HPGD gene exons 1-7 and exon-intron junctions were analysed by direct sequencing. Previously published HPGD- mutated patients were systematically reviewed based on the original clinical description.

Results A novel homozygous c.217+1G>A mutation affecting the obligatory donor splice site of HPGD exon 2 was identified in our proband who showed a mild form of PHO. Review of HPGD-mutated patients outlined all patients manifested digital clubbing, periostosis and acro-osteolysis. (92%), arthralgia (65%) and eczema (33%) were variably associated features. Pachydermia (54%) was mild and mostly limited to palms and sole; cutis vertigis gyrata, blepharoptosis and severe skin thickening were never observed. Besides digital clubbing, PHO infants often presented patent ductus arteriosus (PDA) (32%) and delayed cranial sutures closure (55%).

Conclusion The present findings broaden the allelic spectrum of HPGD gene to include a novel c.217+1G>A mutation. Mutated patients display a homogeneous phenotype mainly consisting in digital clubbing, periostosis, acro-osteolysis, hyperhidrosis and mild pachydermia. Earliest manifestations include delayed closure of the cranial sutures and PDA. In conclusion, the information reported herein would facilitate the diagnosis of PHO due to HPGD mutations.

Key words 15-hydroxyprostaglandindehydrogenase, HPGD, primary hypertrophic osteoarthropathy, digital clubbing, .

Clinical and Experimental Rheumatology 2010; 28: 153-157. HPGD c.217+1G>A splicing mutation in mild PHO / L. Sinibaldi et al.

Lorenzo Sinibaldi, MD, PhD Introduction a distinct homozygous HPGD muta- Ghita Harifi, MD Primary hypertrophic osteoarthropathy tion. Indeed, digital clubbing was the Irene Bottillo, PhD (PHO; OMIM no. 259100) is a heredi- unifying feature in the ten so far report- Miriam Iannicelli, BSc tary systemic disorder characterised by ed HPGD families, while phenotypic Selma El Hassani, MD Francesco Brancati, MD, PhD digital clubbing, arthropathy, periosto- variability was observed with respect Bruno Dallapiccola, MD sis and acro-osteolysis of long bones to cutaneous (mainly pachydermia) and This work was supported by the Italian and cutaneous manifestations including arthropathic manifestations, delayed Ministry of Health (Ricerca Corrente 2009 skin thickening (hence the term pachy- closure of cranial sutures and PDA. to Dr Dallapiccola). dermoperiostosis, PDP) mostly of palms Since no large screening of HPGD gene Please address correspondence to: and soles (1, 2). Additional skin mani- is yet available, report of novel PHO Bruno Dallapiccola, MD, festations include excessive sweating mutated patients may help elucidating CSS-Mendel Institute, and . Three clinical presentations of the HPGD-related phenotype. Viale Regina Margherita 261, PHO are generally recognised: a com- We describe a 13-year-old boy with a 00198 Rome, Italy. plete form characterised by periostosis mild form of PHO harbouring a novel E-mail: [email protected] and pachydermia; an incomplete form homozygous splice-site mutation in Received on July 20, 2009; accepted in with periostosis without pachydermia; HPGD and review the features of the revised form on October 14, 2009. and a ‘forme fruste’ with pachydermia 34 mutated individuals from families © COPYRIGHT CLINICAL AND and minimal or absent skeletal anoma- reported to date. EXPERIMENTAL RHEUMATOLOGY 2010. lies (9, 11). Patent ductus arteriosus (PDA) may be present (6), as well as Clinical report delayed cranial sutures closure prompt- A 13-year-old boy, the second of three ing some author to consider cranio-os- siblings born to first-cousin unaffected teoarthropathy syndrome, or Currarino parents, was referred to our Institute for idiopathic osteoarthropathy (4, 13), as a arthralgia and fingers and toes broad- distinct disorder. Of note, these features ening. No cardiopulmonary or hepatic are clinically indistinguishable from disease was reported nor was apparent those found in PHO phenocopy, the at time of evaluation. Personal history so-called secondary hypertrophic os- revealed polyarthritis since the age of teoarthropathy (SHO), occurring after 5, which was managed as an acute ar- systemic conditions such as pulmonary ticular rheumatism. Progressive fingers and congenital heart diseases (3). and toes deformities associated with Both autosomal dominant and reces- curving of nails and swelling of ankles sive inheritance have been suggested became also apparent from the age of in PHO (OMIM %167100; no. 259100) 5. These features were accompanied (2). Recently, homozygous mutations by pain of legs and forearms with local in the HPGD gene have been identified and hyperhidrosis. in a subset of patients with PHO (12). On clinical examination at 13 years of HPGD encodes the NAD+-dependent age, clubbing of fingers and toes was 15-hydroxyprostaglandin dehydroge- apparent (Fig. 1 a-c) with bilateral re- nase (EC 1.1.1.141), a prostaglandin ducible hyperextension of distal inter- nd E2 (PGE2) catabolising , highly phalangeal articulations of the 2 and expressed in the lung. In patients with 3rd fingers. Maculopapular acne of the HPGD mutations, the loss of enzymatic back was also evident. Skeletal radio- function causes a chronic elevation of graphs of the long bones showed bilat-

circulating PGE2; the consequent pro- eral diaphyseal periostosis and acro- longed peripheral vasodilatation and osteolysis (Fig. 2 a, b). Linear growth

the stimulating effect of PGE2 on os- and psychomotor development were teoblasts and osteoclasts are consistent normal. Blood count showed hypochro- with digital clubbing, acro-osteolysis mic, microcytic, iron-deficient anaemia. and periosteal bone formation observed Calcium and phosphorous serum levels, in PHO patients (12). serum protein electrophoresis, liver and Four PHO families and five additional kidney function assessment were all HPGD mutated kindred were reported within normal ranges. Echocardiogra- so far (8, 12, 14). Moreover, Tariq et al. phy, abdominal ultrasound, chest x-ray (2009) described a large Pakistani fami- and computed tomography scan were ly with isolated congenital clubbing unremarkable. A diagnosis of PHO was Competing interests: none declared. (ICNC; OMIM no. 119900) harbouring then suggested. His arthralgia was re-

154 HPGD c.217+1G>A splicing mutation in mild PHO / L. Sinibaldi et al.

Fig. 1. Hands and feet aspect observed in the patient at 13 years of age with mild clubbing of fingers (a, b) and toes (c). Note “turtle carapace-like” nails particularly evident on the lateral view of the 1st digit (b).

lieved with sodium (75 mg/ day for 9 months). Anaemia recovered after oral iron supplementation therapy. Clinical evaluation of the parents ex- cluded the presence of digital clubbing.

Matherials and methods Peripheral blood samples of proband and parents were collected after obtain- ing an informed consent. Genomic DNA was extracted according to standard procedures and HPGD exons were am- plified in seven PCR fragments (Table I). PCR amplifications were performed with 50 ng of genomic DNA in a 25 μL volume. Exon 1 was amplified in a reaction containing 1X reaction buffer B, 200 μM dNTPs, 0.5 μM of each primer, 3% DMSO and 0.5 U KAPA2G Fast Hot Start DNA polymerase (Kapa Biosystems, Boston, Massachusetts, United States). Thermal cycler condi- tions were 35 cycles of 95°C for 10 sec- onds, 59°C for 10 seconds, and 72°C for 2 seconds, preceeded by 1 minute at 95°C and followed by a final elongation step at 72°C for 30 seconds. PCRs of Fig. 2. Skeletal manifestations consisted of irregular terminal phalanges with acro-osteolysis more exons 2-7 were performed with Ampli evident at the tips of the 2nd, 3rd and 4th terminal phalanges (a) and irregular mild around the diaphyseal portions of the long bones of the forearm. Taq Gold Polymerase (Applied Biosys- tems, Foster City, CA) according to the manufacturer’s instructions at 56°C an- Table I. HPGD gene primers used for genomic amplification and sequencing. nealing temperature. Direct sequencing was performed using BigDye Termina- Exons Forward primer (5’ to 3’) Reverse primer (5’ to 3’) tor v1.1 Cycle Sequencing Kit (PE Ap- 1 GCTGGCTTGACAGTTTCCTC AGTCTCGGAGTGTGTGGGC plied Biosystems, Foster City, CA) on 2 GTGTGTTTATTGTTTGTCCGTC ACGTTCCCAGTTGACAGATTG an automated capillary sequencer (ABI 3130xl, Applied Biosystems). 3 CCTCTCATGGCATAGGACATG GTTTCCATGACTCCAAGAACC Identified single nucleotide substitution 4 GTATTCCTTTTCTCACTTATGC TGAAGATTTGTTTTTGTGGTCC was analysed with two splicing pre- 5 GAGTTTCACAAAGCTATCTGG TGAGATATGACGGTTGTTGTAG diction tools: Human Splicing Finder 6 GAAACTGCTGAAACCTACAAC CTGTATAAGCTTATTTCTTCCC (HSF v.2.4, http://www.umd.be/HSF/) 7 CACATTTCCCTATAACATGTTC AGCTATGGCTAACACATAAGC and NNSPLICE 0.9 (http://www.fruit- fly.org/seq_tools/ splice.html).

155 HPGD c.217+1G>A splicing mutation in mild PHO / L. Sinibaldi et al. % PHO 1 years M 0/1 0/1 55 0/1 32 0/1 33 1/1 28 1/1 67 0/1 54 1/1 92 0/1 33 1/1 63 1/1 100 1/1 100 1/1 100 Yes c.G217+1G>A – this report 3 1 M 1/1 1/1 1/1 0/1 0/1 1/1 1/1 1/1 years 13 Yes Yes N.E. N.E. N.E. N.E. ASD c.418G>C IVS2 –

years 9 3 2 c.1A>T Ex4 SB 1/3 3/3 3/3 2/2 3/0 2/2 3/3 Yes Yes N.E. N.E. N.E. N.E. N.E. N.E. – p.M1L p.M1L p.A140P splice Turkey Turkey Turkey Morocco , 2009 et al. years 23-34 ksel-Konuk - 2 1 1/2 2/2 1/2 0/2 1/1 2/0 1/1 2/2 ü Yes Yes c.418G>C Ex1 N.E. N.E. N.E. N.E. N.E. Y – - 1 2 F 1/1 0/1 1/1 0/1 0/1 0/1 1/1 0/1 1/1 1/1 1/1 1/1 years 21-24 c.120delA c.120delA Ex4 Yes Yes – , 2009 et al. PHO Seifert - 3 1 2/3 1/3 2/3 0/3 0/3 0/3 3/3 0/3 0/3 1/1 2/1 1/1 3/3 Yes Yes c.52G>T Ex2 – 176 Ex1 – years 13 months-11 years 9 - 3 D No 3/3 0/3 0/3 0/3 2/3 3/3 3/3 0/3 2/3 3/3 1/2 3/3 3/3 c.175 – 241 Ex2 – years 13-21 - 2 C 1/2 1/2 2/2 1/2 2/2 2/2 2/2 2/2 2/2 1/1 2/0 1/1 2/2 Yes Yes c.232 – delinsCA delinsCA delCT , 2008 et al. -mutated families. Uppal years 14, 35

- 2 B 2/2 1/2 1/2 0/2 1/2 1/2 2/2 1/2 2/2 1/1 1/1 1/1 2/2 Yes Yes c.418G>C Ex3 – HPGD years 10, 14

- 6 A A 1/6 2/6 0/6 3/6 5/6 3/6 5/6 2/6 4/6 3/3 4/2 3/3 6/6 Yes Yes c.418G>C Ex4 – , 2008 2

- .511T>C .511T>C Ex4 7/4 0/2 0/11 0/11 0/11 0/11 0/11 0/11 0/11 0/11 0/11 0/11 c et al. ICNC – Pakstan Pakistan Pakistan Bangladesh Poland Turkey Netherlands Turkey Tariq Tariq 11 Ex6 Phenotypic and molecular findings in

members velopmental suture closure Other* arteriosus Delayed cranial 0/11 De Patent ductus DNA DNA change Eczema/Flushing 0/11 Acne Seborrhoea Pachydermia Skin Hyperhidrosis 0/11 Swollen joints 0/11 Arthralgia Acro-osteolytis 0/ Sex (F/M) Age or range 2-50 years 14-39 Affected Periostosis

Consanguinity Yes Family ID Table II. Table ICNC: Isolated congenital nail clubbing; PHO: primary hypertrophic osteoarthropathy; F: female; M: male; N.E.: not evaluated. Other associated features found in single ASD:patients; atrial septal defect; SB: saccular bronchiectasiasis. Protein change p.S193P p.A140P p.A140P p.V78QfsX11 p.L59VfsX8 Gly18Cys p.Glu40fsX31 p.A140P Origin Skeletal Digital clubbing 11/11

156 HPGD c.217+1G>A splicing mutation in mild PHO / L. Sinibaldi et al.

Results reported by Tariq et al. (2009) nor in 11 to other HPGD mutations possibly af- HPGD sequence analysis identified in out of 24 HPGD-mutated PHO patients. fecting its function less severely (10). the proband a novel G to A homozygous When present, pachydermia was mild In conclusion, a homogeneous pheno- substitution affecting the obligatory do- and limited to palms and soles while type results from HPGD mutations con- nor splice site of exon 2 (c.217+1G>A). puckering of face and forehead was re- sisting in digital clubbing, periostosis, This change was absent in 150 unaf- ported only in one patient (7). The asso- acro-osteolysis, hyperhidrosis and mild fected individuals and segregated from ciation of HPGD mutations to a rather pachydermia. Large scale screening are heterozygous parents. Bioinformatics mild PHO clinical appearance is also needed to confirm these observations and analysis predicted that the G nucle- supported by the exclusion of mutations to assess the role of HPGD gene among otide at position c.217+1 belonged to in a patient with severe pachydermia of the whole PHO clinical spectrum. the donor splice site of exon 2. In de- hands and feet, facial furrowing with tail, HSF software pointed out that the redundant skin on cheeks and forehead, References c.217+1G>A mutation significantly blepharoptosis, cutis verticis gyrata, se- 1. AUGER M, STAVRIANEAS N: Orphanet Encyclopedia 2004. decreases the consensus value of this borrhoea, and hyperhidrosis 2. CASTORI M, SINIBALDI L, MINGARELLI R, splice site (-27.98%) and NNSPLICE (2). A mild form of pachydermoperios- LACHMAN RS, RIMOIN DL, DALLAPICCOLA indicated the abolition of the donor tosis was recently delineated in which B: Pachydermoperiostosis: an update. Clin splice motif. Both tools predicted the pachydermia was absent or limited to Genet 2005; 68: 477-86. 3. COURY C: Hippocratic fingers and hyper- activation of alternative donor sites in palmar skin (5). Notably, in this family, trophic osteoarthropathy. A study of 350 intron 2. Based on these data, we infer a mutation in the HPGD gene is proba- cases. Br J Dis Chest 1960; 54: 202-9. the c.217+1G>A mutation decreases the ble. However, additional skin manifes- 4. CURRARINO G, TIERNEY RC, GIESEL RG, strength of the consensus exon 2 donor tations including hyperhidrosis (92% WEIHL C: Familial idiopathic osteoarthro- pathy. Am J Roentgenol Radium Ther Nucl site, putting into action an alternative of the cases) and seborrhoea (67%) are Med 1961; 85: 633-44. downstream site. These results point to consistent features of PHO. 5. GIRISHA KM, MANDAL K, PHADKE SR: the possible retention of a share of in- Osteoarticular manifestations are con- Milder form of pachydermoperiostosis: a tron 2. Results of the review of mutated stant findings in HPGD-mutated pa- report of four cases. Clin Dysmorphol 2009; 18: 85-9. HPGD patients are shown in Table II. tients. In particular, digital clubbing of 6. MARTINEZ-LAVIN M, PINEDA C, NAVARRO fingers and toes is observed in all pa- C, BUENDIA A, ZABAL C: Primary hyper- Discussion tients and can manifest since the first trophic osteoarthropathy: another heritable We report on a 13-year-old boy affect- months of life. Periostosis and acro-os- disorder associated with patent ductus arte- riosus. Pediatr Cardiol 1993; 14: 181-2. ed by PHO, homozygous for the novel teolysis have been detected in every ra- 7. OZDEMIR M, YILDIRIM S, MEVLITOGLU I: c.G217+1G>A mutation in the HPGD diologically investigated patient. More En coup de sabre accompanied by pachyder- gene. So far, only seven distinct HPGD than 2/3 of mutated individuals com- moperiostosis: a case report. Clin Exp Rheu- matol 2007; 25: 315-7. pathogenic alterations have been iden- plained of joints pain which notably 8. SEIFERT W, BENINDE J, HOFFMANN K et al.: tified consisting in 4 missense and 3 appeared much earlier than usually re- HPGD mutations cause cranioosteoarthropa- non-sense/frameshift (8,10,12,14). Yet, ported in PHO. Relief of arthralgia by thy but not autosomal dominant digital club- we describe the first splice site muta- nonsteroidal anti-inflammatory therapy bing. Eur J Hum Genet 2009; 17: 1570-6. 9. SINHA GP, CURTIS P, HAIGH D, LEALMAN tion in the gene which is predicted to was observed in treated patients (8, GT, DODDS W, BENNETT CP: Pachydermope- cause the formation of an abnormal present patient). riostosis in childhood. Br J Rheumatol 1997; transcript. Delayed closure of cranial sutures oc- 36: 1224-7. Among previously reported HPGD curs in more than half of the HPGD- 10. TARIQ M, AZEEM Z, ALI G, CHISHTI MS, AHMAD W: Mutation in the HPGD gene mutations, six were identified in single mutated patients while PDA was re- encoding NAD+ dependent 15-hydroxy- families and are distributed in exons 1, ported in about one third of the cases. prostaglandin dehydrogenase underlies iso- 2, 3 and 6, while the p.A140P altera- However, these figures are likely to be lated congenital nail clubbing (ICNC). J Med tion, recurring in 4 out of 11 families, underestimated as these manifestations Genet 2009; 46: 14-20. 11. TOURAINE A, SOLENTE G, GOLE L: Un syn- is located in exon 4 (8, 10, 12, 14). Of may not be evident in adults. Of note, drome osteodermopatique: la pachydermie note, all are homozygous mutations, together with digital clubbing, these plicaturee avec pachyperiostose des extrem- consanguinity being reported in ten out features highlight the juvenile PHO ites. Press Med 1935; 43: 1820-4. of eleven families (Table II). phenotype. 12. UPPAL S, DIGGLE CP, CARR IM et al.: Mutations in 15-hydroxyprostaglandin dehy- Comparison of clinical characteristics No obvious genotype-phenotype cor- drogenase cause primary hypertrophic oste- observed in the 35 HPGD-mutated relation emerges from comparison be- oarthropathy. Nat Genet 2008; 40: 789-93. patients delineates a homogeneous tween HPGD mutation sites and clinical 13. WINTER R, BARAITSER M: London Medical Database, 2005. phenotype defined by digital clubbing, outcome, although the number of mu- 14. YUKSEL-KONUK B, SIRMACI A, AYTEN GE periostosis, acro-osteolysis, which are tated patients is still limited (Table II). et al.: Homozygous mutations in the 15- constant features, and mild skin in- Notably, the p.S193P mutation causes hydroxyprostaglandin dehydrogenase gene volvement especially with respect to isolated congenital nail clubbing. This in patients with primary hypertrophic oste- oarthropathy. Rheumatol Int 2009; Mar 22 pachydermia. In fact, skin thickening alteration localises closer to the C-ter- (Epub ahead of print). was not clinically evident in the family minal domain of the protein compared

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