A novel homozygous missense mutation inFGF23causes Familial ...

2 downloads 0 Views 319KB Size Report
The disease was initially found to result from mutations in GALNT3 encoding a glycosyltransferase. However, more recently, the S71G missense mutation in ...
Hum Genet (2005) 118: 261–266 DOI 10.1007/s00439-005-0026-8

O RI GI N AL IN V ES T IG A T IO N

Ilana Chefetz Æ Raoul Heller Assimina Galli-Tsinopoulou Æ Gabriele Richard Bernd Wollnik Æ Margarita Indelman Friederike Koerber Æ Orit Topaz Æ Reuven Bergman Eli Sprecher Æ Eckhard Schoenau

A novel homozygous missense mutation in FGF23 causes Familial Tumoral Calcinosis associated with disseminated visceral calcification Received: 17 May 2005 / Accepted: 20 June 2005 / Published online: 7 September 2005  Springer-Verlag 2005

Abstract Hyperphosphatemic Familial Tumoral Calcinosis (HFTC; MIM211900) is a rare autosomal recessive disorder characterized by the progressive deposition of calcified masses in cutaneous and subcutaneous tissues, associated with elevated circulating levels of phosphate. The disease was initially found to result from mutations in GALNT3 encoding a glycosyltransferase. However, more recently, the S71G missense mutation in FGF23, encoding a potent phosphaturic protein, was identified in two families. In the present report, we describe a second mutation in FGF23 underlying a severe case

displaying calcifications of cutaneous and numerous extracutaneous tissues. The mutation (M96T) was found to affect a highly conserved methionine residue at position 96 of the protein. These observations illustrate the extent of genetic and phenotypic heterogeneity in HFTC. Key words Calcinosis Æ FGF23 Æ GALNT3 Æ Phosphate Æ Mutation Æ Extraskeletal

Introduction I. Chefetz Æ M. Indelman Æ O. Topaz Æ R. Bergman E. Sprecher (&) Department of Dermatology and Laboratory of Molecular Dermatology, Rambam Medical Center, 9602, Haifa, Israel E-mail: [email protected] Tel.: +972-4-8541919 Fax: +972-4-8542951 I. Chefetz Æ O. Topaz Æ R. Bergman Æ E. Sprecher Bruce Rappaport Faculty of Medicine, Technion–Israel Institute of technology, Haifa, Israel R. Heller Æ B. Wollnik Institute of Human Genetics, University of Cologne, Germany B. Wollnik Center for Molecular Medicine Cologne (CMMC), University of Cologne, Germany A. Galli-Tsinopoulou Fourth Department of Pediatrics, Medical School of Aristotle, University of Thessaloniki, Hospital Papageorgiou, Thessaloniki, Greece G. Richard Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, USA F. Koerber Department of Pediatric Radiology, Children’s Hospital, University of Cologne, Germany E. Schoenau Department of Pediatric Endocrinology, Children’s Hospital, University of Cologne, Germany

Cutaneous calcinosis refers to the ectopic deposition of calcium crystals in the skin (Touart and Sau 1998). Although, acquired calcinosis is a relatively common clinical condition associated with a variety of systemic diseases such as dermatomyositis and chronic renal failure, inherited cutaneous calcinosis, known as familial tumoral calcinosis (FTC; MIM211900), is rare (Polykandriotis et al. 2004). FTC was first described more than a century ago (Giard 1898). It has been mostly reported in Africa and in the Mediterranean basin (McClatchie and Bremner 1969; Metzker et al. 1988). The disease is transmitted in an autosomal recessive manner and has its onset during the first decade of life. It is characterized by the progressive deposition of basic calcium phosphate crystals in juxta-articular skin and soft tissues. Calcifications lead to painful skin ulcerations, secondary skin and bone infections, contractures, which often necessitate surgical removal and result in incapacitating mutilations (Prince et al. 1982; Metzker et al. 1988). Two forms of FTC have been described (Smack et al. 1996). Normophosphatemic FTC (NFTC) is characterized by small tumors located in acral areas, often accompanied by inflammatory manifestations (e.g., conjunctivitis; gingivitis) (Metzker et al. 1988). In hyperphosphatemic FTC (HFTC), elevated levels of phosphate precede the development of tumors, which can weigh more than 1 kg

262

and are usually located over large joints (Prince et al. 1982). The first mutations associated with HFTC were identified in GALNT3 (MIM601756; Topaz et al. 2004; Ichikawa et al. 2005) located on 2q24-2q31, which encodes UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyl-transferase 3 (ppGalNacT3), a Golgi-associated enzyme that mediates mucin type O-linked glycan biosynthesis by instating GalNAc residues on the protein scaffold, targeting serine or threonine residues (Ten Hagen et al. 2003). HFCT causing deleterious mutations in GALNT3 were shown to result in ppGalNacT3 deficiency (Topaz et al. 2005). The nature of ppGalNacT3 physiological substrate(s) is unknown and therefore it is still unclear how ppGalNacT3 deficiency leads to the HFTC phenotype. HFTC represents the metabolic mirror image of autosomal dominant hypophosphatemic rickets (ADHR; MIM307800) caused by gain of function mutations in the fibroblast growth factor 23 (FGF23) gene (MIM605380; ADHR consortium, 2000). FGF23 is part of a group of modulators of phosphate circulating levels known as phosphatonins (Schiavi and Kumar 2004). It has been shown in a number of systems to inhibit phosphate reabsorption in the renal proximal tubules and has therefore been considered for many years as a prime candidate for HFTC (Jan de Beur and Levine 2002). Recently, a homozygous mutation in FGF23 was identified in two HFTC families (Benet-Page`s et al. 2005; Larsson et al. 2005). The sequence change is a missense mutation altering a serine amino acid of the FGF23 sequence. In the present report, we describe a second recessive missense mutation affecting a highly conserved methionine residue, and resulting in HFTC in a young patient of Greek ancestry.

Materials and methods Patient and control individuals Only DNA from the index patient and his mother was available for molecular testing. In addition, we ascertained 104 healthy population-matched individuals as well as an additional 100 healthy individuals of other origins. Blood samples were obtained after having obtained informed and written consent from each participant according to a protocol reviewed and approved by the local Helsinki Committee and by the Israeli Ministry of Health. Fifteen milliliter of blood were drawn from each individual and genomic DNA was isolated from blood samples using the salt chloroform extraction method. Clinical laboratory investigations Measurements of clinical laboratory parameters were performed with commercially available diagnostic kits according to the manufacturer’s instructions. Specifi-

cally, serum alkaline phosphatase (AP) activity was determined kinetically with the IFCC reference method using p-nitrophenylphosphate as substrate (Roche Diagnostics, D-68298 Mannheim), serum parathyroid hormone (PTH) concentrations (intact, biologically active molecule only) were measured using an electrochemiluminescence immunoassay (ECLIA) on a Roche Elecsys 2010 module and serum levels of 1a,25 dihydroxyvitamin D3 were determined using a specific 125I radioimmunoassay after monoclonal immunoextraction (Gamma-B 1,25(OH)D RIA kit from Immunodiagnostics, IDS, D-60329 Frankfurt a.M.) Serum concentration of the C-terminal fragment of the FGF23 molecule was determined using an enzyme-linked immunosorbent assay on a frozen serum sample (ELISA, polyclonal sandwich technique from Immunotopics, San Clemente, CA, USA, Cat.-no. 60-6000). The fractional renal tubular reabsorption of phosphate (TRP) was calculated as [1 – (phosphate clearance/creatinine clearance)]. The renal threshold for phosphate reclamation was derived from the ratio of the maximum rate of renal tubular phosphate reabsorption to glomerular filtration rate (TmaxPO4/GFR) using the standard nomogram of Walton and Bijvoet (1975). Mutational analysis All exons and exon–intron boundaries of the GALNT3 and FGF23 genes were PCR-amplified as previously described (Frishberg et al. 2005; Benet-Page`s et al. 2005). PCR amplification was performed using Taq polymerase (Qiagen, Valencia, CA) and Q solution according to the manufacturer instructions. Gel-purified amplicons were subjected to bi-directional sequencing using Big Dye Terminator (PE Applied Biosystems, Foster City, CA). To verify M96T and to screen the control population, a 210 bp PCR fragment, encompassing exon 2, was PCR-amplified and digested with NlaIII endonuclease.

Results Clinical findings No direct familial relationship between the parents of the index patient could be established, although they both originated from the same Greek village located near the Turkish border. The patient was first seen at 5 years of age for surgical removal of calcified foci from the oral mucosa. Subsequently, he developed large subcutaneous tumors around his wrists, knees (Fig. 1a, b) and ankles (not shown). Small calcified deposits were visible at the external border of the lower eyelids (Fig. 1c). Laboratory parameters (electrolytes, creatinine, urea, uric acid within physiological ranges) did not indicate a decline of renal function over time.

263

Fig. 1 Clinical features of the patient. a Deformation of proximal lower right arm (left panel) and corresponding radiograph (right panel) showing bowing of distal radius and shortened ulna (arrowheads) not related to any fracture, sclerotic narrowing of the bone marrow cavity in the proximal part of radius and ulna, and increased density of the proximal metacarpalia; b Periarticular swelling of right knee area (left panel). The corresponding radiograph (right panel) reveals the absence of recognizable marrow cavity in the distal femoral diaphysis and sclerosis of distal femoral and proximal tibial metaphyses that lack the normally tapered silhouette. There are radiographic signs of subcutaneous calcium deposits on the medial aspect of the knee joint (arrowheads) and vascular calcifications of the deep femoral artery (arrows). c Whitish deposits on lower lid, probably representing calcifications of glands of Moll; d Diffuse but marked increase in echogenicity of medullary pyramids in the kidney reflecting medullary calcinosis. e Quantitative CT of the radius shows reduced trabecular density (–2.21 SD below age-matched normal range; Neu et al. 2001) at the distal radius, corresponding to osteopenia (left panel), and osteosclerosis with undetectable bone marrow cavity in diaphyseal bone (right panel). f Cardiac computer tomography (CT) demonstrating calcified foci in the aortic arch (left panel) and the aortic valve (right panel). Examination was performed with a Multislice-CT (Philips Mx 8000) using the ‘‘Calcium-Score’’-program with reduced mAs (50 – 75 mAs) and 120 kV. We obtained a slice-thickness of 3 mm, collimation 8 · 3, with ultrafast resolution and a 512 · 512 matrix. The rotation time was 0.42 sec, cycle time 0.6 sec. The examination was EKGtriggered and done in breath-holding manner. The anatomical structures are numbered as follows: ascending aorta (1); descending aorta (2); calcification at bottom of aortic arch (3); thymus (4); trachea (5); sternal body (6); vertebral body (7); rib (8); right ventricle (9); left ventricle (10); calcification of aortic valve (11). All pictures were taken between age 12 years and 10 months (radiographs in (a) and (b), and ultrasonography in (c)) and age 13 years and 1 month (photographs in (a) and (b), bone and cardiac CT scans in (e) and (f))

At 13 years and 1 month of age, creatinine clearance was 110 ml/min corrected for body surface (reference: 64–145 ml/min), phosphate clearance was 4.1 ml/min yielding a TRP of 96.27% (normal 82–90%). Correspondingly, the renal threshold phosphate concentration (TmaxPO4/GFR) was markedly elevated with 3.5 mmol/l (normal range: 0.8–1.35 mmol/l). Given the persistent hyperphosphatemia (7.9–8.9 mg/dl, normal range 4– 7 mg/dl) the result for (TmaxPO4/GFR) could only be obtained after multiplying the scales of the Walton–Bijvoet nomogram by a factor of 2.

The patient displayed normocalcemic hypercalciuria (4.56 mg/kg body weight; normal 1,800 RU/ml, normal 5–210 RU/ml). Additional features included sonographic evidence of calcinosis of the renal medullae (Fig. 1d), inhomogeneous bone density assessed by quantitative CT (Neu et al. 2001) (Fig. 1e), and disseminated foci of vascular calcifications including aortic valve and arch (Fig. 1f). Eruption of permanent teeth was delayed with the primary teeth at positions 55, 54, 54, 65, 75, 74, 83, 85 (molars, upper and lower right canines) still in place at 12 years and 4 months. At the same time, disturbed root development with diminished root length was noted for the permanent teeth 16, 26, 36, 46 (1st molars) and 12, 11, 21, 22, 42, 41, 31, 32 (incisors) (not shown, ISO-3950 notation). Consistency and shape of hair and nails were normal. At age 13, the patient’s occipito-frontal head circumference was 54.9 cm (50th–75th centile), his weight was 24.3 kg ( C

264

transversion at position 287 of the FGF23 cDNA sequence (starting from ATG) (Fig. 2a). This mutation was found to be carried in a heterozygous state by the patient’s mother. DNAs from other family members were not available for analysis. Based upon the fact that the mutation abolishes a recognition site for NlaIII, we confirmed the mutation using PCR-RFLP (Fig. 2b). We also excluded the mutation from a pool of 408 chromosomes derived from healthy probands, including 104 population-matched control individuals. The mutation is predicted to result in the substitution of threonine for a methionine residue at position 96 of the FGF23 amino acid sequence. Using the ConSeq server (http://conseq.bioinfo.tau.ac.il), we found that M96 is extremely well-conserved among various homologous proteins in humans and across species (score = 9; range 1–9; calculation performed on 48 unique sequences) (Fig. 2c). To determine the effect of M96T on FGF23 secondary structure, we used the NNPredict software (http://www.cmpharm.ucsf.edu/ ~nomi/nnpredict.html). M96T is predicted to disrupt the 6th antiparallel beta-sheet structure of the protein.

Fig. 2 Mutation analysis. a Direct sequencing reveals an homozygous T fi C transition at position 287 of FGF23 cDNA sequence (upper panel). The wildtype sequence is given for comparision (lower panel). b To confirm 287T>C, a 210 bp fragment encompassing FGF23 exon 2 was PCR-amplified and digested with NlaIII endonuclease. Since 287T>C abolishes a recognition site for NlaIII endonuclease, the patient (P) displays an undigested 210 bp fragment, healthy individuals (C1–C3) display a 148 bp fragment (an additional 62 bp fragment is not visible), and the heterozygous mother of the patient (M) shows both fragment types. (c) ConSeq analysis generates a maximal score for M96 (arrow) of the FGF23 protein sequence. e,b,f,s denote: exposed residue, buried residue, predicted functional residue (highly conserved and exposed) and predicted structural residue (highly conserved and buried), respectively (based upon the calculation of the neuralnetwork algorithm)

Discussion In the present report, we describe a patient with HFTC who was found to carry a novel recessive missense mutation in FGF23. The patient displays elevated 1a,25 dihydroxyvitamin D3 concentrations and increased phosphate reabsorption in the renal tubules resulting in severe hyperphosphatemia. These biochemical features overlap with those described in Fgf23-/- mice (Shimada et al. 2004). The phenotype is consistent with loss of FGF23 activity, since FGF23 normally promotes renal excretion of phosphate, downregulates the anabolic 25-hydroxyvitamin D-1-ahydroxylase and upregulates of the catabolic 24hydroxylase (Shimada et al. 2004; Yamashita et al. 2002 and Segawa et al. 2003). The phenotypic differences between our patient and Fgf23-/- mice are hypercalcemia, elevated alkaline phosphatase, and accumulation of unmineralized osteoid in the mice. These differences may be due to species-specific FGF23 protein function(s) or to the fact that Fgf23-/mice carry biallelic null alleles while our patient carries a recessive missense mutation. To date, 4 mutations in GALNT3 and 1 mutation in FGF23 have been shown to cause HFTC (Table 1). In addition, a founder splice site mutation in GALNT3 was recently shown to underlie an inherited bone disorder, termed hyperostosis-hyperphosphatemia syndrome (HHS), that is characterized by repeated attacks of painful swelling of the long bones and cortical hyperostosis (Frishberg et al. 2005). Thus, HFTC can be caused by recessive mutations in at least two genes, GALNT3 and FGF23; mutations in FGF23 can results in at least two phenotypes, HFTC (recessive loss-offunction mutations) and ADHR (dominant gain-offunction mutations); and mutations in GALNT3 are found in two distinct disorders, HFTC and HHS (Benet-Page`s et al. 2005; Frishberg et al. 2005; Topaz et al.

265 Table 1 Pathogenic mutations detected in HFTC kindreds

Gene

Nucleotide change

Predicted amino acid change

Reference

FGF23 FGF23 FGF23 GALNT3 GALNT3 GALNT3 GALNT3 GALNT3

211A>G 211A>G 287T>C 484C>T 1524 + 1G>A 1524 + 5G>A 484C>T 516-2A>T

S71G S71G M96T R162X K465_Y508del Splicing error R162X Splicing error

Benet-Page`s et al. 2005 Larsson et al. 2005 Present study Topaz et al. 2004 Topaz et al. 2004 Topaz et al. 2004 Ichikawa et al. 2005 Ichikawa et al. 2005

2004; ADHR consortium 2000). This most remarkable instance of combined phenotypic and genetic heterogeneity suggests intersecting or interdependent physiological functions for FGF23 and ppGalNacT3. Since ppGalNacT3 targets serine and threonine residue for O-glycosylation, the fact that the mutation recently described in FGF23 affect a serine residue (Benet-Page`s et al. 2005; Larsson et al. 2005) has been interpreted as suggesting that ppGalNacT3-mediated O-glycosylation may protect normal FGF23 from proteolysis, explaining decreased phosphaturia in HFTC (Larsson et al. 2005). However, protein modeling predicts that Ser71 is unlikely to serve as a target for O-glycosylation (BenetPage`s et al. 2005). In the present study, we report a homozygous recessive mutation in FGF23 altering a methionine residue, suggesting that, at least in the present case, HFTC does not directly result from absent ppGalNacT3-mediated O-glycosylation. We cannot exclude at the present time the possibility that the mutant threonine residue may undergo ppGalNacT3mediated O-glycosylation. However, the NetOGlyc 3.1 software (http://www.cbs.dtu.dk/services/NetOGlyc/) predicts T96 not to be a GalNAc O-glycosylation site. Thus, at present, two other equally plausible scenarios should be considered: ppGalNacT3 may catalyze the post-translational O-glycosylation of a modifier of FGF23 activity or stability; alternatively, aberrant function of ppGalNacT3 or of FGF23 may underlie overlapping phenotypes but affect distinct pathophysiological pathways. Interestingly, although HFTC patients carrying mutations in FGF23 or GALNT3 share many clinical features, the present case as well as two of the three previously reported cases caused by FGF23 mutations (Larsson et al. 2005), display extensive vascular calcifications not observed in HFTC cases associated with GALNT3 mutations (Topaz et al. 2004). Further analysis of genotype-phenotype correlations in additional families as well as in depth dissection of the pathomechanisms underlying FGF23 or ppGalNacT3 dysfunction in HFTC should lead to the delineation of the functional relationship existing between these two proteins. Ackowledgements We acknowledge the family for having participated in the present study. We are grateful to V. Friedman for DNA sequencing services. This study was supported in part by grants provided by The Israeli Ministry of Health-Chief Scientist Office and by the General Trustee Fund.

References ADHR consortium (2000) Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23. Nat Genet 26:345–348 Benet-Page`s A, Orlik P, Strom TM, Lorenz-Depiereux B (2005) An FGF-23 missense mutation causes familial tumoral calcinosis with hyperphosphatemia. Hum Mol Genet 14:385–390 Frishberg Y, Topaz O, Bergman R et al (2005) Identification of a recurrent mutation in GALNT3 demonstrates that hyperostosis-hyperphosphatemia syndrome and familial tumoral calcinosis are allelic disorders. J Mol Med 83:33–38 Giard A (1898) Sur la calcification tibernale. CR Soc Biol 10:1015– 1021 Ichikawa S, Lyles KW, Econs MJ (2005) A novel GALNT3 mutation in a pseudo-autosomal dominant form of tumoral calcinosis: evidence that the disorder is autosomal recessive. J Clin Endocrinol Metab 90:2420–2423 Jan de Beur SM, Levine MA (2002) Molecular pathogenesis of hypophosphatemic rickets. J Clin Endocrinol Metab 87:2467– 2473 Larsson T, Yu X, Davis SI, Draman MS, Mooney SD, Cullen MJ, White KE (2005) A novel recessive mutation in Fibroblast growth factor-23 (FGF23) causes familial tumoral calcinosis. J Clin Endocrinol Metab. 90:2424–2427 McClatchie S, Bremner AD (1969) Tumoural calcinosis–an unrecognized disease. Brit Med J 1:153–155 Metzker A, Eisenstein B, Oren J, Samuel R (1988) Tumoral calcinosis revisited–common and uncommon features. Report of ten cases and review. Eur J Pediatr 147:128–132 Neu CM, Manz F, Rauch F et al (2001) Bone densities and bone size at the distal radius in healthy children and adolescents: a study using peripheral quantitative CT. Bone 28:227–232 Polykandriotis EP, Beutel FK, Horch RE, Grunert J (2004) A case of familial tumoral calcinosis in a neonate and review of the literature. Arch Orthop Trauma Surg 124:563–567 Prince MJ, Schaeffer PC, Goldsmith RS, Chausmer AB (1982) Hyperphosphatemic tumoral calcinosis: association with elevation of serum 1,25-dihydroxycholecalciferol concentrations. Ann Intern Med 96:586–591 Schiavi SC, Kumar R (2004) The phosphatonin pathway: new insights in phosphate homeostasis. Kidney Int 65:1–14 Shimada T, Kakitani M, Yamazaki Y et al (2004) Targeted ablation of FGF23 demonstrates an essential physiological role of FGF23 in phosphate and vitamin D metabolism. J Clin Invest 113:561–568 Smack D, Norton SA, Fitzpatrick JE (1996) Proposal for a pathogenesis-based classification of tumoral calcinosis. Int J Dermatol 35:265–271 Ten Hagen KG, Fritz TA, Tabak LA (2003) All in the family: the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases. Glycobiology 13:1R–16R Topaz O, Bergman R, Mandel U et al (2005) Absence of intraepidermal glycosyltransferase ppGalNAc-T3 expression in familial tumoral calcinosis. Am J Dermatopathol, in press

266 Topaz O, Shurman D, Bergman R et al (2004) Mutations in GALNT3, encoding a protein involved in O-linked glycosylation, cause familial tumoral calcinosis. Nat Genet 36:579– 581 Touart DM, Sau P (1998) Cutaneous deposition diseases. Part II. J Am Acad Dermatol 39:527–544

Walton, Bijvoet (1975) Nomogram for derivation of renal threshold phosphate concentration. Lancet 2(7929):309–310 Yamashita T, Konishi M, Miyake A et al (2002) Fibroblast growth factor (FGF)-23 inhibits renal phosphate reabsorption by activation of the mitogen-activated protein kinase pathway. J Biol Chem 277:28265–28270