J Pediatr Endocr Met 2015; aop
Review article Nurul Nadirah Razali, Ting Tzer Hwu and Karuppiah Thilakavathy*
Phosphate homeostasis and genetic mutations of familial hypophosphatemic rickets Abstract: Hypophosphatemic rickets (HR) is a syndrome of hypophosphatemia and rickets that resembles vitamin D deficiency, which is caused by malfunction of renal tubules in phosphate reabsorption. Phosphate is an essential mineral, which is important for bone and tooth structure. It is regulated by parathyroid hormone, 1,25-dihydroxyvitamin D and fibroblast-growth-factor 23 (FGF23). X-linked hypophosphatemia (XLH), autosomal dominant HR (ADHR), and autosomal recessive HR (ARHR) are examples of hereditary forms of HR, which are mainly caused by mutations in the phosphate regulating endopeptidase homolog, X-linked (PHEX), FGF23, and, dentin matrix protein-1 (DMP1) and ecto-nucleotide pyro phosphatase/phosphodiesterase 1 (ENPP1) genes, respectively. Mutations in these genes are believed to cause elevation of circulating FGF23 protein. Increase in FGF23 disrupts phosphate homeostasis, leading to HR. This review aims to summarize phosphate homeostasis and focuses on the genes and mutations related to XLH, ADHR, and ARHR. A compilation of XLH mutation hotspots based on the PHEX gene database and mutations found in the FGF23, DMP1, and ENPP1 genes are also made available in this review. Keywords: DMP1; ENPP1; FGF23; hypophosphatemia; hypophosphatemic rickets; PHEX; phosphate homeostasis.
*Corresponding author: Assoc. Prof. Dr. Karuppiah Thilakavathy, Clinical Genetics Unit, Department of Obstetrics and Gynaecology, Genetics and Regenerative Medicine Research Centre, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM SERDANG, Selangor Darul Ehsan, Malaysia, Phone: +60389472652, Fax: +60389472646, E-mail: [email protected] Nurul Nadirah Razali: Clinical Genetics Unit, Department of Obstetrics and Gynaecology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, UPM SERDANG, Selangor Darul Ehsan, Malaysia Ting Tzer Hwu: Department of Paediatrics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, UPM SERDANG, Selangor Darul Ehsan, Malaysia.
DOI 10.1515/jpem-2014-0366 Received August 28, 2014; accepted February 26, 2015
Introduction The process of endochondral ossification refers to the transformation of bone from cartilage. Immature and weaker woven bone reabsorbs calcified cartilage matrix before being replaced into stronger and mature lamellar bone (1). During the replacement of woven bone into lamellar bone, osteoid, uncalcified bone matrix is deposited. Deficiency in mineralizing and calcifying of osteoid describes the disorder rickets (1, 2). Derived from an old English word ‘wrick’, which means to twist (3), rickets is a bone disorder that affects the growing skeleton, which causes short stature and lower limb deformities (4). Compared with other stages of human growth, bone grows fastest during childhood. Therefore, it is not surprising that rickets is one of the most well-known disorders among children. Rickets can be caused by several factors, but the most common factor is the lack of vitamin D intake (1, 5). Vitamin D plays a big role in the regulation of calcium and phosphate homeostasis in the human body. Calcium and phosphate are two essential minerals needed by the body to maintain the strength and growth of the bones. Levels of these two minerals in body are regulated by 1,25-dihydroxyvitamin D (1,25(OH)2D) , parathyroid hormone (PTH) and fibroblast-growth-factor 23 (FGF23) (6). Abnormalities of calcium and phosphate regulators will definitely affect the mineralization of osteoid (7). Hypophosphatemic rickets (HR) is a subtype of rickets caused by genetic abnormalities in phosphate regulators. There are genes that may contribute to HR when the genes are mutated; however, the molecular consequences of the expression of the mutated genes are still unclear. Characterized by low phosphate level in the body, HR is considered a rare phosphate metabolism disorder with the rate of occurrence of 1 per 20,000 live births
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 6/19/15 9:13 AM
2 Razali et al.: Phosphate homeostasis and mutations of hypophosphatemic rickets
(2, 8). Low level of phosphate in the body is related to low reabsorption and high excretion of phosphate at the renal tubules in kidney (7, 9). Individuals with HR tend to have growth retardation, short stature, dental abscesses, early tooth loss, bone pain, lower limb deformities, and backache (2, 8, 10, 11). Treatments with phosphate supplement and calcitriol aim at correcting the low blood phosphate level to allow optimal bone growth. Continuous growth hormone uptake may improve growth (12). However, these treatments do not treat the underlying genetic defects that cause the disorder. Recently, Seton and Juppner (13) reported a case wherein a patient who had rickets during childhood with ‘cured’ status acquired the HR disease again at the age of 85. It is not surprising that the disease emerged back after many years as the treatment given did not correct the gene mutation.
Phosphate homeostasis Phosphate is one of the essential nutrients needed by living organisms. Phosphate has many functions, but the most acknowledged function of phosphate is in building and repairing bones and teeth. Phosphate makes up 1% of total body weight, where the majority (85%) can be found in bones and teeth. The remaining 15% exists in other tissues and extracellular fluid (6). Serum phosphate concentration varies with age. Infants have the highest concentration of phosphate, 4.5– 8.3 mg/dL compared with adolescents, 2.5–4.5 mg/dL (14). Infants need more phosphate for growth, which explains why infants have higher phosphate concentration. Bone growth is most rapid during childhood compared with adolescence, during which bones reach its limit and stop growing. Bone structure can be divided into layers; for instance, hypertrophic layer has upper and lower zones. The lower zone will undergo apoptosis at the terminal cells. Apoptosis of epiphyseal chondrocytes helps in new bone regeneration as it removes terminally differentiated cells from cartilage and promotes vascular elements of bone marrow, such as osteoclasts and osteoblasts. Defects in the apoptosis process will lead to rickets (5). However, by restoring phosphorus level in the serum, the process can be rescued because phosphorus is crucial for proper apoptosis of mature chondrocytes in growth plate (6). Reabsorption and excretion of phosphate in the kidney help in maintaining phosphate homeostasis. About 85% of phosphate reabsorption takes place in renal proximal
tubules and the reabsorption is dependent on dietary phosphorus content, PTH, growth hormone (insulin-like growth factor 1) and thyroid hormone concentrations (9).
Regulation of phosphate PTH, FGF23 and 1,25(OH)2D are the major regulators for phosphate homeostasis in the human body (6). PTH is a hormone produced by the parathyroid gland, which mainly helps in elevating calcium concentration. How does PTH secretion relate with phosphate concentration? In general, we comprehend that calcium and phosphate levels are related to each other, in opposite ways. Calcium concentration is decreased if phosphate level is high and vice versa. The interaction between these two minerals helps bone mineralization; at the same time, the level of both phosphate and calcium should not be too high or too low, they must be balanced with each other. To maintain a normal stable phosphate level, increased PTH secretion due to an increase in blood phosphate level will increase phosphate excretion in the urine by renal tubules (Figure 1). Phosphatonins such as FGF23, secreted frizzledrelated protein 4 (sFRP4), fibroblast growth factor 7 (FGF7), and matrix extracellular phosphoglycoprotein (MEPE) have been found to be potential players in the pathogenesis of several hypophosphatemic disorders (15–19). However, FGF23 is the most well-studied circulating humoral phosphaturic factor and is found to be elevated in all familial hypophosphatemic rickets (15, 19, 20). Elevation of FGF23 production affects the regulations of phosphate in kidney and gut. Phosphorus is obtained by cell via secondary active transport, in the form of negatively charged inorganic phosphate (Pi) from extracellular environment. There are three classes of cotransporters of sodium-phosphate, which are expressed in the kidney: type 1, 2, and 3. Type 2 co-transporters, NaPi2a, and NaPi2c are the protein products of SLC34A1 and SLC34A3 genes, respectively, and are responsible for controlling inorganic phosphate reabsorption in the renal proximal tubule (5). FGF23 directly downregulates membrane expression of NaPi-2a by serine phosphorylation of the scaffolding protein Na+/H+ exchange regulatory cofactor (NHERF1) via extracellular signal-regulated kinase 1/2 and serum/glucocorticoid-regulated kinase-1 signaling in proximal tubular segments (21). The suppression of both co-transporters by FGF23 in kidney affects the renal reabsorption of phosphate and causes deficiency of phosphate in blood (22). FGF23 actions require Klotho, a transmembrane protein, as a cofactor (23). Klotho protein
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 6/19/15 9:13 AM
Razali et al.: Phosphate homeostasis and mutations of hypophosphatemic rickets 3
Figure 1: Phosphate regulation by parathyroid hormone (PTH). High level of phosphate stimulates PTH, thus inducing more excretion of phosphate in renal tubular. Meanwhile, in lower state of phosphate level, the excretion of phosphate in renal tubular will be decreased due to less PTH secretion.
binds to multiple FGF receptors, forming a complex that is expressed in the parathyroid gland and expressed predominantly in the distal tubule (24). Klotho-FGF receptors complexes bind to FGF23 with higher affinity compared with FGF receptors alone (25). FGF23-Klotho has been found to suppress PTH secretion by acting directly on the parathyroid gland to enhance phosphaturia and disrupt vitamin D metabolism (26). In the gut, phosphate reabsorption is increased by 1,25(OH)2D. 1-alpha-hydroxylase (CYP27B1) from renal proximal tubule converts 25(OH) D to 1,25(OH)2D. FGF23Klotho reduces the expression and activity levels of CYP27B1 and stimulates 24 hydroxylase, which suppresses the production of 1,25(OH)2D (26). Reduced 1,25(OH)2D will cause decreased reabsorption of phosphate from the gut into the blood stream leading to hypophosphatemia (14).
Genes associated with familial HR PHEX, FGF23, DMP1, and ENPP1 are some of the genes related to familial HR. Gain or loss of functions mutations in these genes can lead to decrease of blood phosphate level via elevation of FGF23 level (Figure 2).
Phosphate-regulating endopeptidase homolog, X-linked (PHEX) PHEX is located on the X chromosome, Xp22.1-p22.2, with the length of 225507 base pairs (27) and comprises of 22
exons, which encode for a 749 amino acid glycoprotein. Mutations in this gene account for about 80% of all familial hypophosphatemia, making it the most common mutated gene among HR patients compared with other genes (3). Based on the PHEX gene database (November 2013), there are about 329 different mutations recorded (28). Mutations in PHEX may cause X-linked dominant HR (XLH), which leads to a defect in reabsorption of phosphate at renal tubules. PHEX helps in bone formation as it encodes for a protein expressed primarily at skeletal level by osteoblasts, osteocytes, and odontoblast (29). However, due to loss-of-function mutation, PHEX loses its own function as it fails to produce functional protein for bone formation, thus stimulating unnecessary proteins, such as FGF23 in bone (30). XLH patients have higher concentration of FGF23 in circulation (3, 31). Various types of mutations are found in the PHEX of HR patients, such as missense, nonsense, insertion, deletion, and splicing site mutations (10, 29). The presence of these mutations may fail to interrupt the process of intermediate cleavage of matrix extracellular glycoprotein (MEPE), a protein involved in biomineralization. Specific cleavage of MEPE generates free protease-resistant ASARM peptides. ASARM peptide is an acidic serine aspartate rich MEPE-associated motif, which has been acknowledged as mineralization inhibitor in bone and teeth, both in in vivo and in vitro studies, where it causes mineralization defects and inhibits renal and intestinal phosphate uptake (32). Thus, interruption in the regulation of phosphate increases phosphate excretion and low secretion of 1,25(OH)2D, which can then lead to hypophosphatemia.
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 6/19/15 9:13 AM
4 Razali et al.: Phosphate homeostasis and mutations of hypophosphatemic rickets
Figure 2: Summary of the most common genes related to familial HR (PHEX, FGF23, and DMP1) and pathophysiology of HR.
Severity of XLH between males and females is believed to be at the same level because of the dominant inheritance. X-linked dominant inheritance is a mode of inheritance that carries dominant gene in chromosome X. Only one copy of allele present is sufficient to cause the disorder. In females, PHEX is subjected to random X inactivation (33); therefore, it is assumed that females should have less severity compared with male. However, it does not necessarily mean that males will be affected more than females because males carry only one X chromosome. If this is the case, does gene dose affect the severity of a disease? Qiu et al. (34) conducted a research on the effect of gene dosage on mineralization defectiveness in Hyp mouse model of XLH, and found that gene dosage gives no effect on mineralization. Thus, it is speculated that there will be no disorder severity differences among different genders (34, 35). Almost half of the reported mutated PHEX results in the production of premature stop codon (X); the signal of translation termination of messenger RNA, and there are almost 100 mutation cases on PHEX that result in stop codon (X) production (28). The expression of premature termination codon produces truncated protein (36). The amino acid that tends to commonly change to premature stop codon in HR patients is arginine (R). The exchange
of amino acid R into X affects bone formation because arginine is originally involved in cell division and releasing of hormones like growth hormone (37). Sabbagh et al. (38) identified C85R, G579R and S711R mutations in XLH patients, and found that the substitution into a large, positively charged residue (R) is likely to affect protein folding. In turn, this leads to the confiscation of mutant proteins inside the cell in contrast to wild-type PHEX which is expressed at the plasma membrane (38). Other amino acid substitutions that can be observed in mutant PHEX are leucine and proline. These are functional amino acids; however, they do not help or enhance bone formation. For example, leucine (L) is produced instead of proline (P) (P534L). Leucine is an aliphatic with the open chain structure; thus, the local hydrophobicity of PHEX products might get affected by its structure (35). Hydrophobicity of the surface is important because it helps in region identification for binding ligand (39). The charge of PHEX is also affected when arginine to proline exchange (R651P) occurs (35). Incorrect protein folding is the consequence of these types of mutations. PHEX is the most studied gene in HR and, therefore, a large variety of different mutations have been found and identified to be the possible cause of loss of function of this gene.
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 6/19/15 9:13 AM
Razali et al.: Phosphate homeostasis and mutations of hypophosphatemic rickets 5
Mutation hotspots of PHEX gene From online PHEX database (28), the list of PHEX mutations (novel and reported mutations) consists of various types of mutations, such as nonsense, missense, deletion, insertion, site splicing, and indels. Certain sites of PHEX exons can be marked as mutation hotspots because several XLH patients have been reported to have the same type of mutations at the sites. Most cases reported missense and nonsense mutations compared with deletion or insertion. Such locations as c.1735, c.58, c.1601, c.2239, c.1699, and c.231 are the most common mutation hotspots in PHEX in HR patients (Table 1). Sato et al. (46) reported that, based on previous molecular analyses, various mutations in PHEX can be found among North Americans, Europeans, African Americans, Saudi Arabians, East Asians, and Indians. Based on Table 1, it is obvious that most of the HR cases reported are of American and European patients. Statistics of HR among East Asians are not as frequent as others; only 15 mutations in PHEX are reported in Korean (47) and Chinese (48) patients, respectively. Meanwhile, for Japanese and Taiwanese, the frequency and distribution of
PHEX are still small in their population as the number of affected patients is limited; thus, conclusions cannot be made yet as further studies are needed (46).
Fibroblast-growth-factor 23 (FGF23) FGF23 is a protein encoded by the FGF23 gene. FGF23 belongs to FGF family that mainly functions in the regulation of cell proliferation and differentiation of FGF receptors (31). FGF23 is associated with autosomal dominant HR (ADHR); it is located in the human chromosome 12p13.3 region and is 18502 base pairs long (27). In terms of main function, FGF23 has similar functions as PHEX, where both genes encode for proteins that express in bone lineage cells involving osteocytes, osteoblasts, and odontoblasts (11). It can be assumed that the expression of FGF23 helps in bone formation and mineralization as well (49). However, mutated FGF23 causes prevention of proteolytic degradation and inactivation of FGF23 production. Due to mutations in FGF23, its production becomes uncontrollable and continues to elevate, leading to hypophosphatemia. This trend is called gain-of-function mutation
Table 1: Summary of mutation hotspot listings on the PHEX in HR patients based on previous case reports. No
Position
Location
Mutation
Amino acid change
Ethnic origin, n
1 2 3 4 5 6 7 8 9
Exon 22 Exon 15 Exon 16 Exon 22 Exon 17 Exon 1 Exon 15 Exon 21 Exon 10
c.*231 c.1601 c.1699 c.2239 c.1735 c.58 c.1645 c.2104 c.1158
A > G C > T
No transcript P534L
American (8) American (1) Korean (1) European (3) Korean (1) American (2) German (3) European (1) Korean (1) Spanish (1) German (1) European (1) American (1) German (2) American (1) Spanish (1) Korean (1) American (2) European (1) Spanish (2) European (1) German (1) Spanish (1) European (1) Finnish (1) European (2)
C > T
C > T
G > A G > C C > T
C > T C > T
G > A
R567X
R747X
G579R G579R R20X
R549X R702X
W386X
n, Indicates number of patients.
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 6/19/15 9:13 AM
Reference (39) (33, 34, 40–42)
(10, 33, 39, 42, 43)
(10, 34, 35, 44, 45)
(29, 34, 39)
(10, 39, 41, 44)
(35, 41) (35, 41, 43)
(41, 42)
6 Razali et al.: Phosphate homeostasis and mutations of hypophosphatemic rickets
where mutation increases the production of proteins from related genes (31). Compared with XLH, ADHR is less common in HR cases as it shows incomplete penetrance (11) and variable time of onset of the clinical phenotype (50). In the case of incomplete penetrance, the traits are autosomal dominant and the disorder phenotypes may or may not be expressed. It might also be affected by other factors, such as lifestyle and environmental factors. The frequency of FGF23 mutation is much less compared to PHEX. From previous studies, there are only four mutations in FGF23 that are known to cause ADHR: R176W (50), R179W (11), R176Q (13), and R179Q (51, 52). Gribaa et al. (51) stated that any mutations located at an 176RXXR179/ S180 destroy the consensus of cleavage site of FGF23, thus interrupting its degradation as the cleavage-resistant protein will remain in active intact form, and eventually exaggerates excretion of phosphate in urine, resulting in hypophosphatemia and low serum 1,25(OH)2D. Table 2 shows the compilation of reported FGF23 mutations.
Dentin matrix protein-1 (DMP1) and ecto-nucleotide pyro phosphatase/ phosphodiesterase 1 (ENPP1) DMP1 and ENPP1 are genes associated with autosomal recessive HR (ARHR), namely, ARHR1 and ARHR2, respectively. The occurrence of HR caused by DMP1 and ENPP1 is quite rare because it is a recessive disorder and fewer mutations are detected in these two genes in HR patients. Located on 4q21-25, DMP1 encodes DMP1 protein and has 21060 base pairs (27). It is a member of Small Intergrin Binding Ligand N-linked Glycoprotein (SIBLING), which is
responsible for bone and dentin mineralization (53) and down regulation of FGF23 (7). DMP1 is expressed highly by osteoblasts and osteocytes in bone lineage, just like PHEX and FGF23 (9). However, due to its loss-of-function mutation, the function of DMP1 is disrupted, causing elevation of FGF23 levels and finally leading to hypophosphatemia. Apart from showing hypophosphatemia state, there is also a sign of osteosclerosis in ARHR patients, particularly at the base of the skull and the calvarial bones (55). Deletion and nucleotide substitution are two most common types of mutations that occur in DMP1; c.485Tdel (E163R) (54), c.1484–1490del, nucleotide substitution of start codon 1A > G (M1V) (19, 55) and c.98, and exchange of nucleotide A > G (W33X) (56). ARHR1 patients manifest hypophosphatemic rickets when 1484–1490del and M1V mutations cause DMP1 to be secreted or not secreted, respectively. A novel mutation was found in Finnish patients at the splice acceptor junction of exon 6 (c.288-1 > A) in DMP1. Heterozygous carriers of the mutation had increased urinary phosphate excretion but showed no obvious effect of skeletal development (60). ENPP1, which has 25 exons and is located on chromosome 6 (6q22-q23), produces ENPP1 protein that plays a role in the regulation of inorganic pyrophosphate (PPi) level, which is an essential physiological inhibitor of calcification (61). The deposition of bone mineral is controlled by the ratio of the concentrations of phosphate to inorganic pyrophosphate and is regulated by ENPP1 (58). Calcium production and accumulation in bone cannot be controlled and inhibited when ENPP1 experiences lossof-function mutation, leading to decrease of phosphate level. Inactivation of ENPP1 has also been described to contribute in generalized arterial calcification of infancy
Table 2: Summary of mutations in the FGF23, DMP1, and ENPP1 genes in HR patients based on previous case reports. Gene
Position
FGF23 DMP1 ENPP1
Exon 3 Exon 3 Exon 2 Exon 6 Exon 6 Exon 2 Exon 3 Exon 6 Intron 2 Intron 5 Exon 5 Exon 24-25 Exon 22 Exon 8 Intron 21
Location
Mutation
Amino acid change
Ethnic origin
Reference
c.526 c.535 c.176 c.485 c.1484-1490 Start codon c.98 c.362 c.55-1 c.288-1 c.247_248 c.2445-798_2778*867 c.2248_2249 c.797 c.2250+1_3
C > T C > T G > A delT Del A > G G > A delC G > C G > A delGG Del insA G > T GTA > CACC
R176W R179W R176Q Truncated Truncated M1V W33X Truncated Splice acceptor Splice acceptor Truncated Truncated Truncated G266V Truncated
Tunisian American American Turkish Lebanese Lebanese Japanese Turkish Spanish Finnish Lebanese Turkish Israeli Arabic Turkish Japanese
(50) (10) (13) (53) (54) (19, 54) (55) (19) (19) (56) (57) (58) (58) (58) (59)
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 6/19/15 9:13 AM
Razali et al.: Phosphate homeostasis and mutations of hypophosphatemic rickets 7
(GACI), a severe autosomal recessive disorder due to the accumulation of calcium in arteries (59). Similar to DMP1, mutations in ENPP1 are very rare compared with others. Lorenz-Depiereux et al. (59) conducted a study on ARHR and found some mutations in ENPP1; homozygous deletion of exons 24 and 25 (c.2445798_2778*867del), insertions of nucleotide A at c.2248_2249, and exchange of nucleotide G > T at c.797 (G266V). In 2011, Saito et al. (57) reported a Japanese HR patient with a novel mutation at the exon-intron junction of exon 21 of ENPP1 (c.2250+1_3 9GTA > CACC). This mutation causes abnormal splicing, which creates a premature stop codon in exon 22. Levy-Litan et al. (61) suggested that mutations in ENPP1 cause ARHR2 via FGF23 pathway. Table 2 shows the compilation of reported DMP1 and ENPP1 mutations.
Conclusion In the human body, phosphate is regulated mainly by parathyroid hormone, FGF23, and 1,25(OH)2D. Impaired phosphate reabsorption in renal proximal tubules leads to X-linked as well as autosomal dominant and autosomal recessive HR. Differential diagnosis of these three types of HR is difficult because they exhibit similar clinical and biochemical features. However, genes that are associated with XLH, ADHR, and ARHR have been found to be different. Thus, screening of PHEX, FGF23, DMP1 and ENPP1 mutations can clinically distinguish these three types of HR patients. Acknowledgments: We would like to thank Associate Professor Dr. Cheah Yoke Kqueen for serving as a peerreviewer for this manuscript before submission. This work is funded by the Fundamental Research Grant Scheme (Grant No. 04-02-13-1327FR) and the MyBrain fellowship by the Ministry of Education, Malaysia. Declaration of interest: The author declares that there is no conflict of interest that could be perceived as prejudicing the impartiality of the review. Funding: This work was supported by the Fundamental Research Grant Scheme (Grant No. 04-02-13-1327FR) funded by the Ministry of Education, Malaysia.
References 1. Wharton B, Bishop N. Rickets. Lancet 2003;362:1389–400. 2. Bhadada SK, Bhansali A, Upreti V, Dutta P, Santosh R, et al. Hypophosphataemic rickets/osteomalacia: a descriptive analysis. Indian J Med Res 2010;131:399–404.
3. Roth KS, Kemp S. Hypophosphatemic Rickets. Medscape; Dec 3, 2014. Available from: http://emedicine.medscape.com/ article/922305-overview. 4. McBride A, Edwards M, Monsell F, Gargan M. Vitamin D-resistant rickets (X-linked hypophosphataemic rickets). Curr Orthop 2007;21:396–9. 5. Tiosano D, Hochberg Z. Hypophosphatemia: the common denominator of all rickets. J Bone Miner Metab 2009;27:392–401. 6. Penido MGMG, Alon US. Phosphate homeostasis and its role in bone health. Pediatr Nephrol 2012;27:2039–48. 7. Jagtap VS, Sarathi V, Lila AR, Bandgar T, Menon P, et al. Hypophosphatemic rickets. Indian J Endocrinol Metab 2012;16:177–82. 8. Douyere D, Joseph C, Gaucher C, Chaussain C, Courson F. Familial hypophosphatemic vitamin D-resistant rickets– prevention of spontaneous dental abscesses on primary teeth: a case report. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;107:525–30. 9. Pettifor JM. What’s new in hypophosphataemic rickets? Eur J Pediatr 2008;167:493–9. 10. Ruppe MD, Brosnan PG, Au KS, Tran PX, Barbara W. Mutation Analysis of PHEX, FGF23 and DMP1 in a cohort of patients with hypophosphatemic rickets. 2012;74:312–8. 11. Kang Q, Xu J, Zhang Z, He J, Lu L, et al. Three novel mutations of the PHEX gene in three Chinese families with X-linked dominant hypophosphatemic rickets. Biochem Biophys Res Commun 2012;81:415–20. 12. Saggese G, Baroncelli GI, Bertelloni S, Perri G. Long-term growth hormone treatment in children with renal hypophosphatemic rickets: effects on growth, mineral metabolism, and bone density. J Pediatr 1995;127:395–402. 13. Seton M, Jüppner H. Autosomal dominant hypophosphatemic rickets in an 85 year old woman: characterization of her disease from infancy through adulthood. Bone 2013;52:640–3. 14. Masi L, Carbonella Sala S, Gozzini A, Pela I, Luzi E, et al. A novel mutation of PHEX causes a dominant X-linked hypophosphatemic rickets. Bone 2007;40. 15. Jonsson K, Zahradnik R, Larsson T, White KE, Sugimoto T, et al. Fibroblast Growth Factor 23 in Oncogenic Osteomalacia and X-Linked Hypophosphatemia. N Engl J Med 2003;348:1656–63. 16. Berndt T, Craig TA, Bowe AE, Vassiliadis J, Reczek D, et al. Secreted frizzled-related protein 4 is a potent tumor-derived phosphaturic agent. J Clin Invest 2003;112:785–94. 17. Carpenter TO, Ellis BK, Insogna KL, Philbrick WM, Sterpka J, et al. FGF7 – an inhibitor of phosphate transport derived from oncogenic osteomalacia-causing tumors. J Clin Endocrinol Metab 2005;90:1012–20. 18. Rowe PS, de Zoysa PA, Dong R, Wang HR, White KE, et al. MEPE, a new gene expressed in bone marrow and tumors causing osteomalacia. Genomics 2000;67:54–68. 19. Feng JQ, Ward LM, Liu S, Lu Y, Xie Y, et al. Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nat Genet 2006;38:1310–5. 20. Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, et al. Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 2012;13:134. 21. Andrukhova O, Zeitz U, Goetz R, Mohammadi M, Lanske B, et al. FGF23 acts directly on renal proximal tubules to induce phosphaturia through activation of the ERK1_2–SGK1 signaling pathway.pdf. Bone 2012;51:621–8.
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 6/19/15 9:13 AM
8 Razali et al.: Phosphate homeostasis and mutations of hypophosphatemic rickets 22. Prié D, Torres PU, Friedlander G. Latest findings in phosphate homeostasis. Kidney Int 2009;75:882–9. 23. Goetz R, Nakada Y, Hu MC, Kurosu H, Wang L, et al. Isolated C-terminal tail of FGF23 alleviates hypophosphatemia by inhibiting FGF23-FGFR-Klotho complex formation. Proc Natl Acad Sci USA 2010;107:407–12. 24. Raina R, Garg G, Sethi SK, Schreiber MJ, Simon JF, et al. Phosphorus Metabolism. J Nephrol Therapeutic 2012; S3:008. 25. Kurosu H, Ogawa Y, Miyoshi M, Yamamoto M, Nandi A, et al. Regulation of fibroblast growth factor-23 signaling by klotho. J Biol Chem 2006;281:6120–3. 26. Ben-dov IZ, Galitzer H, Lavi-moshayoff V, Goetz R, Kuro-o M, et al. The parathyroid is a target organ for FGF23 in rats. J Clin Invest 2007;117:4003–8. 27. Wheeler DL, Barrett T, Benson D a, Bryant SH, Canese K, et al. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res 2007;35(Database issue):D5–12. 28. Sabbagh Y, Jones AO, Tenenhouse HS. PHEXdb, a locus-specific database for mutations causing X-linked hypophosphatemia. Wiley Online Libr 2000;16:1–6. 29. Durmaz E, Zou M, Al-Rijjal RA, Baitei EY, Hammami S, et al. Novel and de novo PHEX mutations in patients with hypophosphatemic rickets. Bone 2013;52:286–91. 30. Nelson AE, Mason RS, Robinson BG. The PEX gene: not a simple answer for X-linked hypophosphataemic rickets and oncogenic osteomalacia. Mol Cell Endocrinol 1997;132:1–5. 31. Fukumoto S, Yamashita T. FGF23 is a hormone-regulating phosphate metabolism–unique biological characteristics of FGF23. Bone 2007;40:1190–5. 32. Rowe PSN. Regulation of bone–renal mineral and energy metabolism: the PHEX, FGF23, DMP1, MEPE ASARM pathway. Crit Rev Eukaryot Gene Expr 2013;22:61–86. 33. Song H-R, Park J-W, Cho D-Y, Yang JH, Yoon H-R, et al. PHEX gene mutations and genotype-phenotype analysis of Korean patients with hypophosphatemic rickets. J Korean Med Sci 2007;22:981. 34. Qiu Z, Travers R, Rauch F, Glorieux F, Scriver C, et al. Effect of gene dose and parental origin on bone histomorphometry in X-linked Hyp mice. Bone 2004;34:134–9. 35. Francis F, Strom TM, Hennig S, Bo A, Lorenz B, et al. Genomic organization of the human PEX gene mutated in X-linked genomic organization of the human PEX gene mutated in X-linked dominant hypophosphatemic rickets. Genome Res 1997;7:573–85. 36. Morey M, Castro-Feijóo L, Barreiro J, Cabanas P, Pombo M, et al. Genetic diagnosis of X-linked dominant hypophosphatemic rickets in a cohort study: tubular reabsorption of phosphate and 1,25(OH)2D serum levels are associated with PHEX mutation type. BMC Med Genet 2011;12:116. 37. Reichardt B, Schrader M, Mojto J, Mehltretter G, Muller O-A, et al. The decrease in growth hormone (GH) Response after Repeated Stimulation with GH-releasing hormone os partly caused by an elevation of somatostatin tonus. J Clin Endocrinol Metab1996;81:1994–8. 38. Sabbagh Y, Boileau G, DesGroseillers L, Tenenhouse HS. Disease-causing missense mutations in the PHEX gene interfere with membrane targeting of the recombinant protein. Hum Mol Genet 2001;10:1539–46. 39. Young L, Jernigan RL, Covell DG. A role for surface hydrophobicity in protein-protein recognition. Protein Sci 1994;3:717–29.
40. Ichikawa S, Traxler E a, Estwick S a, Curry LR, Johnson ML, et al. Mutational survey of the PHEX gene in patients with X-linked hypophosphatemic rickets. Bone 2008;43:663–6. 41. Holm IA, Nelson AE, Robinson BG, Mason RS, Marsh DJ, et al. Mutational analysis and genotype-phenotype correlation of the PHEX gene in X-linked hypophosphatemic rickets. J Clin Endocrinol Metab 2001;86:3889–99. 42. Lo F-S, Kuo M-T, Wang C-J, Chang C-H, Lee Z-L, et al. Two novel PHEX mutations in Taiwanese patients with X-linked hypophosphatemic rickets. Nephron Physiol 2006;103:157–63. 43. Quinlan C, Guegan K, Offiah A, Neill RO, Hiorns MP, et al. Growth in PHEX-associated X-linked hypophosphatemic rickets: the importance of early treatment. Pediatr Nephrol 2012;27:581–8. 44. Živičnjak M, Schnabel D, Staude H, Even G, Marx M, et al. Three-year growth hormone treatment in short children with X-linked hypophosphatemic rickets: effects on linear growth and body disproportion. J Clin Endocrinol Metab 2011;96:E2097–105. 45. Cho HY, Lee BH, Kang JH, Ha IS, Cheong HI, et al. A clinical and molecular genetic study of hypophosphatemic rickets in children. Pediatr Res 2005;58:329–33. 46. Sato K, Tajima T, Nakae J, Adachi M, Asakura Y, et al. Three novel PHEX gene mutations in Japanese patients with X-linked hypophosphatemic rickets. Pediatr Res 2000;48:536–40. 47. Kim J, Yang KH, Nam JS, Choi JR, Song J, et al. A novel PHEX mutation in a Korean patient with sporadic hypophosphatemic rickets. Ann Clin Lab Sci 2009;39:182–7. 48. Yang L, Yang J, Huang X. PHEX gene mutation in a Chinese family with six cases of X linked hypophosphatemic rickets. J Pediatr Endocrinol Metab 2013;26:4–5. 49. Dunstan CR, Zhou H, Seibel MJ. Fibroblast growth factor 23: a phosphatonin regulating phosphate homeostasis? Endocrinology 2004;145:3084–6. 50. Imel EA, Peacock M, Gray AK, Padgett LR, Hui SL, et al. Iron modifies plasma FGF23 differently in autosomal dominant hypophosphatemic rickets and healthy humans. J Clin Endocrinol Metab 2011;96:3541–9. 51. Gribaa M, Younes M, Bouyacoub Y, Korbaa W, Ben Charfeddine I, et al. An autosomal dominant hypophosphatemic rickets phenotype in a Tunisian family caused by a new FGF23 missense mutation. J Bone Miner Metab 2010;28:111–5. 52. Kinoshita Y, Saito T, Shimizu Y, Hori M, Taguchi M, et al. Mutational analysis of patients with FGF23-related hypophosphatemic rickets. Eur J Endocrinol 2012;167:165–72. 53. Farrow EG, Davis SI, Ward LM, Summers LJ, Bubbear JS, et al. Molecular analysis of DMP1 mutants causing autosomal recessive hypophosphatemic rickets. Bone 2009;44:287–94. 54. Turan S, Aydin C, Bereket A, Akcay T, Güran T, et al. Identification of a novel dentin matrix protein-1 (DMP-1) mutation and dental anomalies in a kindred with autosomal recessive hypophosphatemia. Bone 2010;46:402–9. 55. Lorenz-Depiereux B, Benet-Pages A, Eckstein G, TenenbaumRakover Y, Wagenstaller J, et al. Hereditary hypophosphatemic rickets with hypercalciuria is caused by mutations in the sodium-phosphate cotransporter gene SLC34A3. Am J Hum Genet 2006;78:193–201. 56. Koshida R, Yamaguchi H, Yamasaki K, Tsuchimochi W, Yonekawa T, et al. A novel nonsense mutation in the DMP1
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 6/19/15 9:13 AM
Razali et al.: Phosphate homeostasis and mutations of hypophosphatemic rickets 9
gene in a Japanese family with autosomal recessive hypophosphatemic rickets. J Bone Miner Metab 2010;28:585–90. 57. Saito T, Shimizu Y, Hori M, Taguchi M, Igarashi T, et al. A patient with hypophosphatemic rickets and ossification of posterior longitudinal ligament caused by a novel homozygous mutation in ENPP1 gene. Bone. Elsevier Inc. 2011;49:913–6. 58. Mackenzie NCW, Zhu D, Milne EM, van ’t Hof R, Martin A, et al. Altered bone development and an increase in FGF-23 expression in Enpp1(-/-) mice. PLoS One 2012;7:e32177. 59. Lorenz-Depiereux B, Schnabel D, Tiosano D, Häusler G, Strom TM. Loss-of-function ENPP1 mutations cause both
generalized arterial calcification of infancy and autosomalrecessive hypophosphatemic rickets. Am J Hum Genet 2010;86:267–72. 60. Mäkitie O, Pereira RC, Kaitila I, Turan S, Bastepe M, et al. Long-term clinical outcome and carrier phenotype in autosomal recessive hypophosphatemia caused by a novel DMP1 mutation. J Bone Miner Res 2010;25:2165–74. 61. Levy-Litan V, Hershkovitz E, Avizov L, Leventhal N, Bercovich D, et al. Autosomal-recessive hypophosphatemic rickets is associated with an inactivation mutation in the ENPP1 gene. Am J Hum Genet 2010;86:273–8.
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 6/19/15 9:13 AM
Review article Nurul Nadirah Razali, Ting Tzer Hwu and Karuppiah Thilakavathy*
Phosphate homeostasis and genetic mutations of familial hypophosphatemic rickets Abstract: Hypophosphatemic rickets (HR) is a syndrome of hypophosphatemia and rickets that resembles vitamin D deficiency, which is caused by malfunction of renal tubules in phosphate reabsorption. Phosphate is an essential mineral, which is important for bone and tooth structure. It is regulated by parathyroid hormone, 1,25-dihydroxyvitamin D and fibroblast-growth-factor 23 (FGF23). X-linked hypophosphatemia (XLH), autosomal dominant HR (ADHR), and autosomal recessive HR (ARHR) are examples of hereditary forms of HR, which are mainly caused by mutations in the phosphate regulating endopeptidase homolog, X-linked (PHEX), FGF23, and, dentin matrix protein-1 (DMP1) and ecto-nucleotide pyro phosphatase/phosphodiesterase 1 (ENPP1) genes, respectively. Mutations in these genes are believed to cause elevation of circulating FGF23 protein. Increase in FGF23 disrupts phosphate homeostasis, leading to HR. This review aims to summarize phosphate homeostasis and focuses on the genes and mutations related to XLH, ADHR, and ARHR. A compilation of XLH mutation hotspots based on the PHEX gene database and mutations found in the FGF23, DMP1, and ENPP1 genes are also made available in this review. Keywords: DMP1; ENPP1; FGF23; hypophosphatemia; hypophosphatemic rickets; PHEX; phosphate homeostasis.
*Corresponding author: Assoc. Prof. Dr. Karuppiah Thilakavathy, Clinical Genetics Unit, Department of Obstetrics and Gynaecology, Genetics and Regenerative Medicine Research Centre, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM SERDANG, Selangor Darul Ehsan, Malaysia, Phone: +60389472652, Fax: +60389472646, E-mail: [email protected] Nurul Nadirah Razali: Clinical Genetics Unit, Department of Obstetrics and Gynaecology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, UPM SERDANG, Selangor Darul Ehsan, Malaysia Ting Tzer Hwu: Department of Paediatrics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, UPM SERDANG, Selangor Darul Ehsan, Malaysia.
DOI 10.1515/jpem-2014-0366 Received August 28, 2014; accepted February 26, 2015
Introduction The process of endochondral ossification refers to the transformation of bone from cartilage. Immature and weaker woven bone reabsorbs calcified cartilage matrix before being replaced into stronger and mature lamellar bone (1). During the replacement of woven bone into lamellar bone, osteoid, uncalcified bone matrix is deposited. Deficiency in mineralizing and calcifying of osteoid describes the disorder rickets (1, 2). Derived from an old English word ‘wrick’, which means to twist (3), rickets is a bone disorder that affects the growing skeleton, which causes short stature and lower limb deformities (4). Compared with other stages of human growth, bone grows fastest during childhood. Therefore, it is not surprising that rickets is one of the most well-known disorders among children. Rickets can be caused by several factors, but the most common factor is the lack of vitamin D intake (1, 5). Vitamin D plays a big role in the regulation of calcium and phosphate homeostasis in the human body. Calcium and phosphate are two essential minerals needed by the body to maintain the strength and growth of the bones. Levels of these two minerals in body are regulated by 1,25-dihydroxyvitamin D (1,25(OH)2D) , parathyroid hormone (PTH) and fibroblast-growth-factor 23 (FGF23) (6). Abnormalities of calcium and phosphate regulators will definitely affect the mineralization of osteoid (7). Hypophosphatemic rickets (HR) is a subtype of rickets caused by genetic abnormalities in phosphate regulators. There are genes that may contribute to HR when the genes are mutated; however, the molecular consequences of the expression of the mutated genes are still unclear. Characterized by low phosphate level in the body, HR is considered a rare phosphate metabolism disorder with the rate of occurrence of 1 per 20,000 live births
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 6/19/15 9:13 AM
2 Razali et al.: Phosphate homeostasis and mutations of hypophosphatemic rickets
(2, 8). Low level of phosphate in the body is related to low reabsorption and high excretion of phosphate at the renal tubules in kidney (7, 9). Individuals with HR tend to have growth retardation, short stature, dental abscesses, early tooth loss, bone pain, lower limb deformities, and backache (2, 8, 10, 11). Treatments with phosphate supplement and calcitriol aim at correcting the low blood phosphate level to allow optimal bone growth. Continuous growth hormone uptake may improve growth (12). However, these treatments do not treat the underlying genetic defects that cause the disorder. Recently, Seton and Juppner (13) reported a case wherein a patient who had rickets during childhood with ‘cured’ status acquired the HR disease again at the age of 85. It is not surprising that the disease emerged back after many years as the treatment given did not correct the gene mutation.
Phosphate homeostasis Phosphate is one of the essential nutrients needed by living organisms. Phosphate has many functions, but the most acknowledged function of phosphate is in building and repairing bones and teeth. Phosphate makes up 1% of total body weight, where the majority (85%) can be found in bones and teeth. The remaining 15% exists in other tissues and extracellular fluid (6). Serum phosphate concentration varies with age. Infants have the highest concentration of phosphate, 4.5– 8.3 mg/dL compared with adolescents, 2.5–4.5 mg/dL (14). Infants need more phosphate for growth, which explains why infants have higher phosphate concentration. Bone growth is most rapid during childhood compared with adolescence, during which bones reach its limit and stop growing. Bone structure can be divided into layers; for instance, hypertrophic layer has upper and lower zones. The lower zone will undergo apoptosis at the terminal cells. Apoptosis of epiphyseal chondrocytes helps in new bone regeneration as it removes terminally differentiated cells from cartilage and promotes vascular elements of bone marrow, such as osteoclasts and osteoblasts. Defects in the apoptosis process will lead to rickets (5). However, by restoring phosphorus level in the serum, the process can be rescued because phosphorus is crucial for proper apoptosis of mature chondrocytes in growth plate (6). Reabsorption and excretion of phosphate in the kidney help in maintaining phosphate homeostasis. About 85% of phosphate reabsorption takes place in renal proximal
tubules and the reabsorption is dependent on dietary phosphorus content, PTH, growth hormone (insulin-like growth factor 1) and thyroid hormone concentrations (9).
Regulation of phosphate PTH, FGF23 and 1,25(OH)2D are the major regulators for phosphate homeostasis in the human body (6). PTH is a hormone produced by the parathyroid gland, which mainly helps in elevating calcium concentration. How does PTH secretion relate with phosphate concentration? In general, we comprehend that calcium and phosphate levels are related to each other, in opposite ways. Calcium concentration is decreased if phosphate level is high and vice versa. The interaction between these two minerals helps bone mineralization; at the same time, the level of both phosphate and calcium should not be too high or too low, they must be balanced with each other. To maintain a normal stable phosphate level, increased PTH secretion due to an increase in blood phosphate level will increase phosphate excretion in the urine by renal tubules (Figure 1). Phosphatonins such as FGF23, secreted frizzledrelated protein 4 (sFRP4), fibroblast growth factor 7 (FGF7), and matrix extracellular phosphoglycoprotein (MEPE) have been found to be potential players in the pathogenesis of several hypophosphatemic disorders (15–19). However, FGF23 is the most well-studied circulating humoral phosphaturic factor and is found to be elevated in all familial hypophosphatemic rickets (15, 19, 20). Elevation of FGF23 production affects the regulations of phosphate in kidney and gut. Phosphorus is obtained by cell via secondary active transport, in the form of negatively charged inorganic phosphate (Pi) from extracellular environment. There are three classes of cotransporters of sodium-phosphate, which are expressed in the kidney: type 1, 2, and 3. Type 2 co-transporters, NaPi2a, and NaPi2c are the protein products of SLC34A1 and SLC34A3 genes, respectively, and are responsible for controlling inorganic phosphate reabsorption in the renal proximal tubule (5). FGF23 directly downregulates membrane expression of NaPi-2a by serine phosphorylation of the scaffolding protein Na+/H+ exchange regulatory cofactor (NHERF1) via extracellular signal-regulated kinase 1/2 and serum/glucocorticoid-regulated kinase-1 signaling in proximal tubular segments (21). The suppression of both co-transporters by FGF23 in kidney affects the renal reabsorption of phosphate and causes deficiency of phosphate in blood (22). FGF23 actions require Klotho, a transmembrane protein, as a cofactor (23). Klotho protein
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 6/19/15 9:13 AM
Razali et al.: Phosphate homeostasis and mutations of hypophosphatemic rickets 3
Figure 1: Phosphate regulation by parathyroid hormone (PTH). High level of phosphate stimulates PTH, thus inducing more excretion of phosphate in renal tubular. Meanwhile, in lower state of phosphate level, the excretion of phosphate in renal tubular will be decreased due to less PTH secretion.
binds to multiple FGF receptors, forming a complex that is expressed in the parathyroid gland and expressed predominantly in the distal tubule (24). Klotho-FGF receptors complexes bind to FGF23 with higher affinity compared with FGF receptors alone (25). FGF23-Klotho has been found to suppress PTH secretion by acting directly on the parathyroid gland to enhance phosphaturia and disrupt vitamin D metabolism (26). In the gut, phosphate reabsorption is increased by 1,25(OH)2D. 1-alpha-hydroxylase (CYP27B1) from renal proximal tubule converts 25(OH) D to 1,25(OH)2D. FGF23Klotho reduces the expression and activity levels of CYP27B1 and stimulates 24 hydroxylase, which suppresses the production of 1,25(OH)2D (26). Reduced 1,25(OH)2D will cause decreased reabsorption of phosphate from the gut into the blood stream leading to hypophosphatemia (14).
Genes associated with familial HR PHEX, FGF23, DMP1, and ENPP1 are some of the genes related to familial HR. Gain or loss of functions mutations in these genes can lead to decrease of blood phosphate level via elevation of FGF23 level (Figure 2).
Phosphate-regulating endopeptidase homolog, X-linked (PHEX) PHEX is located on the X chromosome, Xp22.1-p22.2, with the length of 225507 base pairs (27) and comprises of 22
exons, which encode for a 749 amino acid glycoprotein. Mutations in this gene account for about 80% of all familial hypophosphatemia, making it the most common mutated gene among HR patients compared with other genes (3). Based on the PHEX gene database (November 2013), there are about 329 different mutations recorded (28). Mutations in PHEX may cause X-linked dominant HR (XLH), which leads to a defect in reabsorption of phosphate at renal tubules. PHEX helps in bone formation as it encodes for a protein expressed primarily at skeletal level by osteoblasts, osteocytes, and odontoblast (29). However, due to loss-of-function mutation, PHEX loses its own function as it fails to produce functional protein for bone formation, thus stimulating unnecessary proteins, such as FGF23 in bone (30). XLH patients have higher concentration of FGF23 in circulation (3, 31). Various types of mutations are found in the PHEX of HR patients, such as missense, nonsense, insertion, deletion, and splicing site mutations (10, 29). The presence of these mutations may fail to interrupt the process of intermediate cleavage of matrix extracellular glycoprotein (MEPE), a protein involved in biomineralization. Specific cleavage of MEPE generates free protease-resistant ASARM peptides. ASARM peptide is an acidic serine aspartate rich MEPE-associated motif, which has been acknowledged as mineralization inhibitor in bone and teeth, both in in vivo and in vitro studies, where it causes mineralization defects and inhibits renal and intestinal phosphate uptake (32). Thus, interruption in the regulation of phosphate increases phosphate excretion and low secretion of 1,25(OH)2D, which can then lead to hypophosphatemia.
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 6/19/15 9:13 AM
4 Razali et al.: Phosphate homeostasis and mutations of hypophosphatemic rickets
Figure 2: Summary of the most common genes related to familial HR (PHEX, FGF23, and DMP1) and pathophysiology of HR.
Severity of XLH between males and females is believed to be at the same level because of the dominant inheritance. X-linked dominant inheritance is a mode of inheritance that carries dominant gene in chromosome X. Only one copy of allele present is sufficient to cause the disorder. In females, PHEX is subjected to random X inactivation (33); therefore, it is assumed that females should have less severity compared with male. However, it does not necessarily mean that males will be affected more than females because males carry only one X chromosome. If this is the case, does gene dose affect the severity of a disease? Qiu et al. (34) conducted a research on the effect of gene dosage on mineralization defectiveness in Hyp mouse model of XLH, and found that gene dosage gives no effect on mineralization. Thus, it is speculated that there will be no disorder severity differences among different genders (34, 35). Almost half of the reported mutated PHEX results in the production of premature stop codon (X); the signal of translation termination of messenger RNA, and there are almost 100 mutation cases on PHEX that result in stop codon (X) production (28). The expression of premature termination codon produces truncated protein (36). The amino acid that tends to commonly change to premature stop codon in HR patients is arginine (R). The exchange
of amino acid R into X affects bone formation because arginine is originally involved in cell division and releasing of hormones like growth hormone (37). Sabbagh et al. (38) identified C85R, G579R and S711R mutations in XLH patients, and found that the substitution into a large, positively charged residue (R) is likely to affect protein folding. In turn, this leads to the confiscation of mutant proteins inside the cell in contrast to wild-type PHEX which is expressed at the plasma membrane (38). Other amino acid substitutions that can be observed in mutant PHEX are leucine and proline. These are functional amino acids; however, they do not help or enhance bone formation. For example, leucine (L) is produced instead of proline (P) (P534L). Leucine is an aliphatic with the open chain structure; thus, the local hydrophobicity of PHEX products might get affected by its structure (35). Hydrophobicity of the surface is important because it helps in region identification for binding ligand (39). The charge of PHEX is also affected when arginine to proline exchange (R651P) occurs (35). Incorrect protein folding is the consequence of these types of mutations. PHEX is the most studied gene in HR and, therefore, a large variety of different mutations have been found and identified to be the possible cause of loss of function of this gene.
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 6/19/15 9:13 AM
Razali et al.: Phosphate homeostasis and mutations of hypophosphatemic rickets 5
Mutation hotspots of PHEX gene From online PHEX database (28), the list of PHEX mutations (novel and reported mutations) consists of various types of mutations, such as nonsense, missense, deletion, insertion, site splicing, and indels. Certain sites of PHEX exons can be marked as mutation hotspots because several XLH patients have been reported to have the same type of mutations at the sites. Most cases reported missense and nonsense mutations compared with deletion or insertion. Such locations as c.1735, c.58, c.1601, c.2239, c.1699, and c.231 are the most common mutation hotspots in PHEX in HR patients (Table 1). Sato et al. (46) reported that, based on previous molecular analyses, various mutations in PHEX can be found among North Americans, Europeans, African Americans, Saudi Arabians, East Asians, and Indians. Based on Table 1, it is obvious that most of the HR cases reported are of American and European patients. Statistics of HR among East Asians are not as frequent as others; only 15 mutations in PHEX are reported in Korean (47) and Chinese (48) patients, respectively. Meanwhile, for Japanese and Taiwanese, the frequency and distribution of
PHEX are still small in their population as the number of affected patients is limited; thus, conclusions cannot be made yet as further studies are needed (46).
Fibroblast-growth-factor 23 (FGF23) FGF23 is a protein encoded by the FGF23 gene. FGF23 belongs to FGF family that mainly functions in the regulation of cell proliferation and differentiation of FGF receptors (31). FGF23 is associated with autosomal dominant HR (ADHR); it is located in the human chromosome 12p13.3 region and is 18502 base pairs long (27). In terms of main function, FGF23 has similar functions as PHEX, where both genes encode for proteins that express in bone lineage cells involving osteocytes, osteoblasts, and odontoblasts (11). It can be assumed that the expression of FGF23 helps in bone formation and mineralization as well (49). However, mutated FGF23 causes prevention of proteolytic degradation and inactivation of FGF23 production. Due to mutations in FGF23, its production becomes uncontrollable and continues to elevate, leading to hypophosphatemia. This trend is called gain-of-function mutation
Table 1: Summary of mutation hotspot listings on the PHEX in HR patients based on previous case reports. No
Position
Location
Mutation
Amino acid change
Ethnic origin, n
1 2 3 4 5 6 7 8 9
Exon 22 Exon 15 Exon 16 Exon 22 Exon 17 Exon 1 Exon 15 Exon 21 Exon 10
c.*231 c.1601 c.1699 c.2239 c.1735 c.58 c.1645 c.2104 c.1158
A > G C > T
No transcript P534L
American (8) American (1) Korean (1) European (3) Korean (1) American (2) German (3) European (1) Korean (1) Spanish (1) German (1) European (1) American (1) German (2) American (1) Spanish (1) Korean (1) American (2) European (1) Spanish (2) European (1) German (1) Spanish (1) European (1) Finnish (1) European (2)
C > T
C > T
G > A G > C C > T
C > T C > T
G > A
R567X
R747X
G579R G579R R20X
R549X R702X
W386X
n, Indicates number of patients.
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 6/19/15 9:13 AM
Reference (39) (33, 34, 40–42)
(10, 33, 39, 42, 43)
(10, 34, 35, 44, 45)
(29, 34, 39)
(10, 39, 41, 44)
(35, 41) (35, 41, 43)
(41, 42)
6 Razali et al.: Phosphate homeostasis and mutations of hypophosphatemic rickets
where mutation increases the production of proteins from related genes (31). Compared with XLH, ADHR is less common in HR cases as it shows incomplete penetrance (11) and variable time of onset of the clinical phenotype (50). In the case of incomplete penetrance, the traits are autosomal dominant and the disorder phenotypes may or may not be expressed. It might also be affected by other factors, such as lifestyle and environmental factors. The frequency of FGF23 mutation is much less compared to PHEX. From previous studies, there are only four mutations in FGF23 that are known to cause ADHR: R176W (50), R179W (11), R176Q (13), and R179Q (51, 52). Gribaa et al. (51) stated that any mutations located at an 176RXXR179/ S180 destroy the consensus of cleavage site of FGF23, thus interrupting its degradation as the cleavage-resistant protein will remain in active intact form, and eventually exaggerates excretion of phosphate in urine, resulting in hypophosphatemia and low serum 1,25(OH)2D. Table 2 shows the compilation of reported FGF23 mutations.
Dentin matrix protein-1 (DMP1) and ecto-nucleotide pyro phosphatase/ phosphodiesterase 1 (ENPP1) DMP1 and ENPP1 are genes associated with autosomal recessive HR (ARHR), namely, ARHR1 and ARHR2, respectively. The occurrence of HR caused by DMP1 and ENPP1 is quite rare because it is a recessive disorder and fewer mutations are detected in these two genes in HR patients. Located on 4q21-25, DMP1 encodes DMP1 protein and has 21060 base pairs (27). It is a member of Small Intergrin Binding Ligand N-linked Glycoprotein (SIBLING), which is
responsible for bone and dentin mineralization (53) and down regulation of FGF23 (7). DMP1 is expressed highly by osteoblasts and osteocytes in bone lineage, just like PHEX and FGF23 (9). However, due to its loss-of-function mutation, the function of DMP1 is disrupted, causing elevation of FGF23 levels and finally leading to hypophosphatemia. Apart from showing hypophosphatemia state, there is also a sign of osteosclerosis in ARHR patients, particularly at the base of the skull and the calvarial bones (55). Deletion and nucleotide substitution are two most common types of mutations that occur in DMP1; c.485Tdel (E163R) (54), c.1484–1490del, nucleotide substitution of start codon 1A > G (M1V) (19, 55) and c.98, and exchange of nucleotide A > G (W33X) (56). ARHR1 patients manifest hypophosphatemic rickets when 1484–1490del and M1V mutations cause DMP1 to be secreted or not secreted, respectively. A novel mutation was found in Finnish patients at the splice acceptor junction of exon 6 (c.288-1 > A) in DMP1. Heterozygous carriers of the mutation had increased urinary phosphate excretion but showed no obvious effect of skeletal development (60). ENPP1, which has 25 exons and is located on chromosome 6 (6q22-q23), produces ENPP1 protein that plays a role in the regulation of inorganic pyrophosphate (PPi) level, which is an essential physiological inhibitor of calcification (61). The deposition of bone mineral is controlled by the ratio of the concentrations of phosphate to inorganic pyrophosphate and is regulated by ENPP1 (58). Calcium production and accumulation in bone cannot be controlled and inhibited when ENPP1 experiences lossof-function mutation, leading to decrease of phosphate level. Inactivation of ENPP1 has also been described to contribute in generalized arterial calcification of infancy
Table 2: Summary of mutations in the FGF23, DMP1, and ENPP1 genes in HR patients based on previous case reports. Gene
Position
FGF23 DMP1 ENPP1
Exon 3 Exon 3 Exon 2 Exon 6 Exon 6 Exon 2 Exon 3 Exon 6 Intron 2 Intron 5 Exon 5 Exon 24-25 Exon 22 Exon 8 Intron 21
Location
Mutation
Amino acid change
Ethnic origin
Reference
c.526 c.535 c.176 c.485 c.1484-1490 Start codon c.98 c.362 c.55-1 c.288-1 c.247_248 c.2445-798_2778*867 c.2248_2249 c.797 c.2250+1_3
C > T C > T G > A delT Del A > G G > A delC G > C G > A delGG Del insA G > T GTA > CACC
R176W R179W R176Q Truncated Truncated M1V W33X Truncated Splice acceptor Splice acceptor Truncated Truncated Truncated G266V Truncated
Tunisian American American Turkish Lebanese Lebanese Japanese Turkish Spanish Finnish Lebanese Turkish Israeli Arabic Turkish Japanese
(50) (10) (13) (53) (54) (19, 54) (55) (19) (19) (56) (57) (58) (58) (58) (59)
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 6/19/15 9:13 AM
Razali et al.: Phosphate homeostasis and mutations of hypophosphatemic rickets 7
(GACI), a severe autosomal recessive disorder due to the accumulation of calcium in arteries (59). Similar to DMP1, mutations in ENPP1 are very rare compared with others. Lorenz-Depiereux et al. (59) conducted a study on ARHR and found some mutations in ENPP1; homozygous deletion of exons 24 and 25 (c.2445798_2778*867del), insertions of nucleotide A at c.2248_2249, and exchange of nucleotide G > T at c.797 (G266V). In 2011, Saito et al. (57) reported a Japanese HR patient with a novel mutation at the exon-intron junction of exon 21 of ENPP1 (c.2250+1_3 9GTA > CACC). This mutation causes abnormal splicing, which creates a premature stop codon in exon 22. Levy-Litan et al. (61) suggested that mutations in ENPP1 cause ARHR2 via FGF23 pathway. Table 2 shows the compilation of reported DMP1 and ENPP1 mutations.
Conclusion In the human body, phosphate is regulated mainly by parathyroid hormone, FGF23, and 1,25(OH)2D. Impaired phosphate reabsorption in renal proximal tubules leads to X-linked as well as autosomal dominant and autosomal recessive HR. Differential diagnosis of these three types of HR is difficult because they exhibit similar clinical and biochemical features. However, genes that are associated with XLH, ADHR, and ARHR have been found to be different. Thus, screening of PHEX, FGF23, DMP1 and ENPP1 mutations can clinically distinguish these three types of HR patients. Acknowledgments: We would like to thank Associate Professor Dr. Cheah Yoke Kqueen for serving as a peerreviewer for this manuscript before submission. This work is funded by the Fundamental Research Grant Scheme (Grant No. 04-02-13-1327FR) and the MyBrain fellowship by the Ministry of Education, Malaysia. Declaration of interest: The author declares that there is no conflict of interest that could be perceived as prejudicing the impartiality of the review. Funding: This work was supported by the Fundamental Research Grant Scheme (Grant No. 04-02-13-1327FR) funded by the Ministry of Education, Malaysia.
References 1. Wharton B, Bishop N. Rickets. Lancet 2003;362:1389–400. 2. Bhadada SK, Bhansali A, Upreti V, Dutta P, Santosh R, et al. Hypophosphataemic rickets/osteomalacia: a descriptive analysis. Indian J Med Res 2010;131:399–404.
3. Roth KS, Kemp S. Hypophosphatemic Rickets. Medscape; Dec 3, 2014. Available from: http://emedicine.medscape.com/ article/922305-overview. 4. McBride A, Edwards M, Monsell F, Gargan M. Vitamin D-resistant rickets (X-linked hypophosphataemic rickets). Curr Orthop 2007;21:396–9. 5. Tiosano D, Hochberg Z. Hypophosphatemia: the common denominator of all rickets. J Bone Miner Metab 2009;27:392–401. 6. Penido MGMG, Alon US. Phosphate homeostasis and its role in bone health. Pediatr Nephrol 2012;27:2039–48. 7. Jagtap VS, Sarathi V, Lila AR, Bandgar T, Menon P, et al. Hypophosphatemic rickets. Indian J Endocrinol Metab 2012;16:177–82. 8. Douyere D, Joseph C, Gaucher C, Chaussain C, Courson F. Familial hypophosphatemic vitamin D-resistant rickets– prevention of spontaneous dental abscesses on primary teeth: a case report. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;107:525–30. 9. Pettifor JM. What’s new in hypophosphataemic rickets? Eur J Pediatr 2008;167:493–9. 10. Ruppe MD, Brosnan PG, Au KS, Tran PX, Barbara W. Mutation Analysis of PHEX, FGF23 and DMP1 in a cohort of patients with hypophosphatemic rickets. 2012;74:312–8. 11. Kang Q, Xu J, Zhang Z, He J, Lu L, et al. Three novel mutations of the PHEX gene in three Chinese families with X-linked dominant hypophosphatemic rickets. Biochem Biophys Res Commun 2012;81:415–20. 12. Saggese G, Baroncelli GI, Bertelloni S, Perri G. Long-term growth hormone treatment in children with renal hypophosphatemic rickets: effects on growth, mineral metabolism, and bone density. J Pediatr 1995;127:395–402. 13. Seton M, Jüppner H. Autosomal dominant hypophosphatemic rickets in an 85 year old woman: characterization of her disease from infancy through adulthood. Bone 2013;52:640–3. 14. Masi L, Carbonella Sala S, Gozzini A, Pela I, Luzi E, et al. A novel mutation of PHEX causes a dominant X-linked hypophosphatemic rickets. Bone 2007;40. 15. Jonsson K, Zahradnik R, Larsson T, White KE, Sugimoto T, et al. Fibroblast Growth Factor 23 in Oncogenic Osteomalacia and X-Linked Hypophosphatemia. N Engl J Med 2003;348:1656–63. 16. Berndt T, Craig TA, Bowe AE, Vassiliadis J, Reczek D, et al. Secreted frizzled-related protein 4 is a potent tumor-derived phosphaturic agent. J Clin Invest 2003;112:785–94. 17. Carpenter TO, Ellis BK, Insogna KL, Philbrick WM, Sterpka J, et al. FGF7 – an inhibitor of phosphate transport derived from oncogenic osteomalacia-causing tumors. J Clin Endocrinol Metab 2005;90:1012–20. 18. Rowe PS, de Zoysa PA, Dong R, Wang HR, White KE, et al. MEPE, a new gene expressed in bone marrow and tumors causing osteomalacia. Genomics 2000;67:54–68. 19. Feng JQ, Ward LM, Liu S, Lu Y, Xie Y, et al. Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nat Genet 2006;38:1310–5. 20. Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, et al. Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 2012;13:134. 21. Andrukhova O, Zeitz U, Goetz R, Mohammadi M, Lanske B, et al. FGF23 acts directly on renal proximal tubules to induce phosphaturia through activation of the ERK1_2–SGK1 signaling pathway.pdf. Bone 2012;51:621–8.
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 6/19/15 9:13 AM
8 Razali et al.: Phosphate homeostasis and mutations of hypophosphatemic rickets 22. Prié D, Torres PU, Friedlander G. Latest findings in phosphate homeostasis. Kidney Int 2009;75:882–9. 23. Goetz R, Nakada Y, Hu MC, Kurosu H, Wang L, et al. Isolated C-terminal tail of FGF23 alleviates hypophosphatemia by inhibiting FGF23-FGFR-Klotho complex formation. Proc Natl Acad Sci USA 2010;107:407–12. 24. Raina R, Garg G, Sethi SK, Schreiber MJ, Simon JF, et al. Phosphorus Metabolism. J Nephrol Therapeutic 2012; S3:008. 25. Kurosu H, Ogawa Y, Miyoshi M, Yamamoto M, Nandi A, et al. Regulation of fibroblast growth factor-23 signaling by klotho. J Biol Chem 2006;281:6120–3. 26. Ben-dov IZ, Galitzer H, Lavi-moshayoff V, Goetz R, Kuro-o M, et al. The parathyroid is a target organ for FGF23 in rats. J Clin Invest 2007;117:4003–8. 27. Wheeler DL, Barrett T, Benson D a, Bryant SH, Canese K, et al. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res 2007;35(Database issue):D5–12. 28. Sabbagh Y, Jones AO, Tenenhouse HS. PHEXdb, a locus-specific database for mutations causing X-linked hypophosphatemia. Wiley Online Libr 2000;16:1–6. 29. Durmaz E, Zou M, Al-Rijjal RA, Baitei EY, Hammami S, et al. Novel and de novo PHEX mutations in patients with hypophosphatemic rickets. Bone 2013;52:286–91. 30. Nelson AE, Mason RS, Robinson BG. The PEX gene: not a simple answer for X-linked hypophosphataemic rickets and oncogenic osteomalacia. Mol Cell Endocrinol 1997;132:1–5. 31. Fukumoto S, Yamashita T. FGF23 is a hormone-regulating phosphate metabolism–unique biological characteristics of FGF23. Bone 2007;40:1190–5. 32. Rowe PSN. Regulation of bone–renal mineral and energy metabolism: the PHEX, FGF23, DMP1, MEPE ASARM pathway. Crit Rev Eukaryot Gene Expr 2013;22:61–86. 33. Song H-R, Park J-W, Cho D-Y, Yang JH, Yoon H-R, et al. PHEX gene mutations and genotype-phenotype analysis of Korean patients with hypophosphatemic rickets. J Korean Med Sci 2007;22:981. 34. Qiu Z, Travers R, Rauch F, Glorieux F, Scriver C, et al. Effect of gene dose and parental origin on bone histomorphometry in X-linked Hyp mice. Bone 2004;34:134–9. 35. Francis F, Strom TM, Hennig S, Bo A, Lorenz B, et al. Genomic organization of the human PEX gene mutated in X-linked genomic organization of the human PEX gene mutated in X-linked dominant hypophosphatemic rickets. Genome Res 1997;7:573–85. 36. Morey M, Castro-Feijóo L, Barreiro J, Cabanas P, Pombo M, et al. Genetic diagnosis of X-linked dominant hypophosphatemic rickets in a cohort study: tubular reabsorption of phosphate and 1,25(OH)2D serum levels are associated with PHEX mutation type. BMC Med Genet 2011;12:116. 37. Reichardt B, Schrader M, Mojto J, Mehltretter G, Muller O-A, et al. The decrease in growth hormone (GH) Response after Repeated Stimulation with GH-releasing hormone os partly caused by an elevation of somatostatin tonus. J Clin Endocrinol Metab1996;81:1994–8. 38. Sabbagh Y, Boileau G, DesGroseillers L, Tenenhouse HS. Disease-causing missense mutations in the PHEX gene interfere with membrane targeting of the recombinant protein. Hum Mol Genet 2001;10:1539–46. 39. Young L, Jernigan RL, Covell DG. A role for surface hydrophobicity in protein-protein recognition. Protein Sci 1994;3:717–29.
40. Ichikawa S, Traxler E a, Estwick S a, Curry LR, Johnson ML, et al. Mutational survey of the PHEX gene in patients with X-linked hypophosphatemic rickets. Bone 2008;43:663–6. 41. Holm IA, Nelson AE, Robinson BG, Mason RS, Marsh DJ, et al. Mutational analysis and genotype-phenotype correlation of the PHEX gene in X-linked hypophosphatemic rickets. J Clin Endocrinol Metab 2001;86:3889–99. 42. Lo F-S, Kuo M-T, Wang C-J, Chang C-H, Lee Z-L, et al. Two novel PHEX mutations in Taiwanese patients with X-linked hypophosphatemic rickets. Nephron Physiol 2006;103:157–63. 43. Quinlan C, Guegan K, Offiah A, Neill RO, Hiorns MP, et al. Growth in PHEX-associated X-linked hypophosphatemic rickets: the importance of early treatment. Pediatr Nephrol 2012;27:581–8. 44. Živičnjak M, Schnabel D, Staude H, Even G, Marx M, et al. Three-year growth hormone treatment in short children with X-linked hypophosphatemic rickets: effects on linear growth and body disproportion. J Clin Endocrinol Metab 2011;96:E2097–105. 45. Cho HY, Lee BH, Kang JH, Ha IS, Cheong HI, et al. A clinical and molecular genetic study of hypophosphatemic rickets in children. Pediatr Res 2005;58:329–33. 46. Sato K, Tajima T, Nakae J, Adachi M, Asakura Y, et al. Three novel PHEX gene mutations in Japanese patients with X-linked hypophosphatemic rickets. Pediatr Res 2000;48:536–40. 47. Kim J, Yang KH, Nam JS, Choi JR, Song J, et al. A novel PHEX mutation in a Korean patient with sporadic hypophosphatemic rickets. Ann Clin Lab Sci 2009;39:182–7. 48. Yang L, Yang J, Huang X. PHEX gene mutation in a Chinese family with six cases of X linked hypophosphatemic rickets. J Pediatr Endocrinol Metab 2013;26:4–5. 49. Dunstan CR, Zhou H, Seibel MJ. Fibroblast growth factor 23: a phosphatonin regulating phosphate homeostasis? Endocrinology 2004;145:3084–6. 50. Imel EA, Peacock M, Gray AK, Padgett LR, Hui SL, et al. Iron modifies plasma FGF23 differently in autosomal dominant hypophosphatemic rickets and healthy humans. J Clin Endocrinol Metab 2011;96:3541–9. 51. Gribaa M, Younes M, Bouyacoub Y, Korbaa W, Ben Charfeddine I, et al. An autosomal dominant hypophosphatemic rickets phenotype in a Tunisian family caused by a new FGF23 missense mutation. J Bone Miner Metab 2010;28:111–5. 52. Kinoshita Y, Saito T, Shimizu Y, Hori M, Taguchi M, et al. Mutational analysis of patients with FGF23-related hypophosphatemic rickets. Eur J Endocrinol 2012;167:165–72. 53. Farrow EG, Davis SI, Ward LM, Summers LJ, Bubbear JS, et al. Molecular analysis of DMP1 mutants causing autosomal recessive hypophosphatemic rickets. Bone 2009;44:287–94. 54. Turan S, Aydin C, Bereket A, Akcay T, Güran T, et al. Identification of a novel dentin matrix protein-1 (DMP-1) mutation and dental anomalies in a kindred with autosomal recessive hypophosphatemia. Bone 2010;46:402–9. 55. Lorenz-Depiereux B, Benet-Pages A, Eckstein G, TenenbaumRakover Y, Wagenstaller J, et al. Hereditary hypophosphatemic rickets with hypercalciuria is caused by mutations in the sodium-phosphate cotransporter gene SLC34A3. Am J Hum Genet 2006;78:193–201. 56. Koshida R, Yamaguchi H, Yamasaki K, Tsuchimochi W, Yonekawa T, et al. A novel nonsense mutation in the DMP1
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 6/19/15 9:13 AM
Razali et al.: Phosphate homeostasis and mutations of hypophosphatemic rickets 9
gene in a Japanese family with autosomal recessive hypophosphatemic rickets. J Bone Miner Metab 2010;28:585–90. 57. Saito T, Shimizu Y, Hori M, Taguchi M, Igarashi T, et al. A patient with hypophosphatemic rickets and ossification of posterior longitudinal ligament caused by a novel homozygous mutation in ENPP1 gene. Bone. Elsevier Inc. 2011;49:913–6. 58. Mackenzie NCW, Zhu D, Milne EM, van ’t Hof R, Martin A, et al. Altered bone development and an increase in FGF-23 expression in Enpp1(-/-) mice. PLoS One 2012;7:e32177. 59. Lorenz-Depiereux B, Schnabel D, Tiosano D, Häusler G, Strom TM. Loss-of-function ENPP1 mutations cause both
generalized arterial calcification of infancy and autosomalrecessive hypophosphatemic rickets. Am J Hum Genet 2010;86:267–72. 60. Mäkitie O, Pereira RC, Kaitila I, Turan S, Bastepe M, et al. Long-term clinical outcome and carrier phenotype in autosomal recessive hypophosphatemia caused by a novel DMP1 mutation. J Bone Miner Res 2010;25:2165–74. 61. Levy-Litan V, Hershkovitz E, Avizov L, Leventhal N, Bercovich D, et al. Autosomal-recessive hypophosphatemic rickets is associated with an inactivation mutation in the ENPP1 gene. Am J Hum Genet 2010;86:273–8.
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 6/19/15 9:13 AM
