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Abstract: Digital clubbing is the most prominent feature in primary (PHO) and secondary (pulmonary) hypertrophic osteoarthropathy (HO). Homozygous and compound heterozygous germline mutations in the 15-hydroxyprostaglandin dehydrogenase (HPGD) gene encoding the major prostaglandin PGE2 catabolizing enzyme have been recently described in familial PHO cases. Elevated prostaglandin levels in affected individuals with cytokine-mediated tissue remodelling and vascular stimulation may underlie PHO and associated features as hyperhidrosis, acroosteolysis, pachyderma, periostosis and arthritis. We present clinical and biochemical data of three unrelated PHO families with HPGD mutations. The truncating mutation c.175_176del was found in two of our families and in one of the recently described pedigrees, all of European origin. We present evidence that c.175_176del is a recurrent mutation rather than an ancient founder allele. Two novel heterozygous HPGD mutations, the nonsense mutation c.118G>T (p.Glu40X) and the missense mutation c.563C>T (p.Thr188Ile), could be identified in a third family. We postulate that all HPGD mutations constitute loss-of-function alleles due to protein truncation or missense changes that affect hydrogen bonds lining the 15-PGDH enzyme reaction cavity. Elevated prostaglandin levels may give rise to use of non-steroidal anti-inflammatory drugs; however, therapeutic strategies have not been reported to date. Naproxen treatment in one of our mutation-positive patients resulted in alleviation of pain caused by periostosis and arthritis as well as reduction in substantially elevated prostaglandin levels, while no significant effects on digital clubbing, hyperhidrosis and pachyderma were observed. Further experience with nonsteroidal anti-inflammatory drugs in PHO is awaited.
Digital clubbing due to enlargement of the distal phalanges and resulting in overcurvature of the nails is a classical sign in medicine with heterogeneous aetiology. It often indicates chronic hypoxia in advanced bronchopulmonary disorders, thoracic tumors or cardiovascular diseases. Less frequent causes include gastrointestinal, endocrine, haematological and hereditary conditions. However, the pathogenesis of the hypertrophy of the soft tissue with increased vascularity is not well understood.
By deciphering rare hereditary disorders, the understanding of common diseases may be essentially enhanced with tremendous impact on the development of new therapeutic options. Recently, mutations in the HPGD gene [MIM 601688] encoding the major prostaglandin PGE2 catabolizing enzyme have been reported to cause primary hypertrophic osteoarthropathy (PHO) [MIM 259100] whose most prominent feature is digital clubbing (1–4). Other early onset developmental anomalies in PHO patients include delayed closure of the cranial sutures and patent ductus arteriosus. Intriguingly, PHO individuals share most of their findings with patients that display hypertrophic osteoarthropathy (HO) secondary to systemic, often pulmonary disease. In addition to digital clubbing, these are fibroblast stimulation, glandular hypertrophy and osteoblast proliferation, which cause thickening of the skin, bothersome palmoplantar hyperhidrosis and joint disease (‘pachydermoperiostosis’). Acroosteolysis of the distal phalanges of the fingers and toes due to osteoclast stimulation is also commonly observed (5,6). Although promotional influences of prostaglandins and prostaglandin analogues on the hair follicle are obvious by virtue of clinical observations and experimental data, hair growth and hair structure are usually not affected in PHO (7).
A crucial role of the prostaglandin metabolism in the pathogene-sis of PHO was first anticipated by clinical studies that reported major HO features in patients receiving long-term prostaglandin therapy (7–11). By positional cloning in consanguineous families with several affected individuals, Uppal et al. identified mutations in the HPGD gene on chromosome 4q34 (1). Seven coding HPGD exons encode the 266 amino acid 15-hydroxyprostaglandin dehydrogenase, which is ubiquitously expressed. It constitutes the main enzyme of prostaglandin degradation whose enzyme activity is NAD+ dependent.
In this study, we performed clinical and molecular analyses in individuals of three unrelated families with PHO. Major findings are summarized in Table 1. The parents of patient 1239 were first cousins once removed and of German origin (Fig. 1). A common ancestor and distant parental consanguinity was not known but could also be postulated in the Austrian family 1293 in which four of ten children were affected with PHO (Fig. 1). All patients were of normal intelligence and height and did not suffer from pulmonary disease. Blood samples were collected from all patients and family members after informed consent, and genomic DNA was extracted using standard procedures. We performed direct sequencing of the coding region and adjacent exon-intron boundaries of the HPGD gene on an ABI PRISM 3130 genetic analyser (Applied Biosystems, Darmstadt, Germany). Primer sequences are available on request. Patients of families 1239 and 1293 were both shown to carry the homozygous 2-basepair deletion c.175_176del (p.Leu59fs) in exon 2 (Fig. 1) that has already been described in the Polish family published by Uppal et al. (1). While we cannot rule out the possibility of a common ancestral origin of one of our families with the Polish pedigree, we can practically exclude an ancient founder allele in families 1239 and 1293. The known non-coding single nucleotide polymorphism IVS4 + 39C>T (rs3775977, http://genome.ucsc.edu) was found on both parental alleles from the propositus of family 1293, whereas the index patient of family 1239 carries the wildtype C allele homozygously (data not shown). However, as this SNP is rather common it cannot be determined with certainty whether the former allele represents a mutated founder allele with an additional mutational event or a completely different allele which seems to be more likely. Hence, we consider c.175_176del rather a recurrent mutation than a common founder allele.
Table 1. Clinical and molecular features of PHO patients
|Current age (years)||18||14||65|
|Clubbing of fingers and toes||√||√||√|
|Arthralgia of large joints||√||∅||∅|
|Delayed closure of cranial sutures||√||∅||∅|
|Patent ductus arteriosus||∅||∅||∅|
|Urinary PGE2 levels (ng/mmol creatinine) (healthy controls <50)||235||186||n.d.|
|HPGD genotype||c.118G>T (p.Glu40X) c.563C>T (p.Thr188Ile)||c.175_176del (p.Leu59fs) c.175_176del (p.Leu59fs)||c.175_176del (p.Leu59fs) c.175_176del (p.Leu59fs)|
Figure 1. Top left: Pedigrees of families 1239 and 1293. Bottom left: Electropherograms depicting the heterozygous and homozygous 2-basepair deletion c.175_176del (p.Leu59fs) in exon 2 of the HPGD gene identified in the above families (shown are the reverse sequences for reasons of clarity). Right: Propositus of family 1239 at the age of 8 and 14 years.
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Two novel HPGD mutations could be identified in family 1234 (Fig. 2). On the paternal allele, the heterozygous nonsense mutation c.118G>T (p.Glu40X) was found. On the maternal allele, we detected the missense mutation c.563C>T (p.Thr188Ile) that affects an evolutionarily conserved residue and was not present among controls. Consistent with the two recently described missense mutations c.418G>C (p.Ala140Pro) and c.577T>C (p.Ser193Pro) and evidence from the HPGD/15-PGDH crystal structure(1), threonine T188 is also part of a network of hydrogen bonds that line the enzyme reaction cavity of 15-PGDH.
Figure 2. Top left: Pedigree of family 1234 and part of the HPGD/15-PGDH crystal structure, demonstrating that threonine p.Thr188 and the amino acids affected by the two recently described missense mutations c.418G>C (p.Ala140Pro) and c.577T>C (p.Ser193Pro) are part of a network of hydrogen bonds lining the reaction cavity (from Uppal et al.) (1). Bottom left: Two novel HPGD mutations could be identified in the index patient. Electropherograms depicting the paternally inherited heterozygous nonsense mutation c.118G>T (p.Glu40X) in exon 2, and the maternally inherited heterozygous missense mutation c.563C>T (p.Thr188Ile) in exon 6. Top right: Phenotype of the index patient featuring paw-like hands with digital clubbing and overcurvature of the finger nails, palmar keratosis with accentuated furrowing, and tubular shape of lower legs caused by periostosis and soft tissue enlargement. Bottom right: In silico characterisation of the novel missense mutation c.563C>T (p.Thr188Ile) in exon 6 of the HPGD gene identified on the maternal allele of the propositus of family 1234. Multiple protein sequence alignment, generated with ClustalW, showing that the identified HPGD missense mutation affects an amino acid that is highly conserved through evolution. The affected threonine residue (p.Thr188) is indicated.
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So far, all published HPGD mutations represent protein truncating alleles or affect residues that are thought to be indispensable for 15-PGDH enzyme activity. Thus, we postulate that HPGD mutations leading to autosomal recessive PHO consistently constitute loss-of-function alleles. Rather uncommon for recessive disorders and especially for recessive loss-of-function alleles, minor clinical features in terms of moderate thickening and furrowing of both palms with palmoplantar hyperhidrosis were also present in the heterozygous father of family 1239. However, clubbing, bone and joint pathologies were absent. Although elevated moderately in inflammatory diseases or digital clubbing secondary to pulmonary diseases, all of our patients demonstrated significantly elevated urinary prostaglandin PGE2 levels thereby offering a screening test for individuals suggestive of PHO. Additionally, patients with palmoplantar hyperhidrosis – presenting with an isolated symptom of PHO – did not indicate raised urinary prostaglandin PGE2 levels (46.8 ± 15.5 ng/mmol creatinine, n = 7, controls <50, n = 5).
Loss of 15-PGDH activity can be observed in a number of different tumors with increasing evidence that HPGD is a tumor suppressor gene (12–14). Secondary HO is a well-known consequence of certain neoplasias and a common pathogenetic mechanism may underlie primary and secondary forms of HO. However, tumor predisposition has not been reported in families with PHO so far. One of our patients (F1239) showed massive thymus hyperplasia with multiple cysts at the age of 13 years most probably as a result of chronic inflammation. Answering the question if PHO patients bear an increased risk for tumors is of paramount importance for their management and surveillance, but currently there is no convincing evidence for this hypothesis.
The delineation of the molecular basis of PHO offers therapeutic options such as application of cyclooxygenase (COX) inhibitors. A 3-month treatment course with the non-steroidal anti-inflammatory drug naproxen in one of our severely affected PHO patients led to symptomatic relief of painful periostosis and arthritis, and to marked reduction in urinary prostaglandine PGE2 levels (235 to 64 ng/mmol creatinine), but had no effect on long-standing clinical signs. Thus, other cytokines may be involved in evoking the diverse clinical traits of PHO. Alternatively and more probably, treatment with prostanoid antagonists has to be started before cytokine-mediated remodelling and anatomical changes in diverse tissues commence.