2. What is HR ( Hypophosphatemic Rickets ) ?
HR including …
Hypo : means low or less and a few amount of
something
Phosphate (Pi) : it is one of vital elements in the body
Rickets : is a softening of bones in children due to
deficiency or impaired metabolism of vitamin
D, Phosphorus or Calcium, potentially leading to
fractures and deformity. Rickets is among the most
frequent childhood diseases in many developing
countries.

3. Then :
HR means lack of Phosphate (Pi) that
leads to Rickets.
Phosphate is the major stimulus for
1 α Hydroxylase.

4. Phosphate metabolism
Phosphates are absorbed from foodstuff. Specialized channel proteins called sodium-
phosphate transporters 2b (or NaPi2b) located at the surface of the epithelial cells of the
small intestine are performing the task. About 1.5 g of phosphate are captured daily by
this process by a normal adult.

5. Once in the bloodstream, phosphates can be absorbed by organs and tissues. A
part of it is stored in bones.
Then, phosphates reach the kidneys where most of it is filtered out of the blood.
But before being eliminated in the urine, another channel protein (NaPi2a)
similar to the one that capture phosphate from foodstuff bring it back to
bloodstream in a process called 'reabsorption'. This steps of filtration and
reabsorption taking place in the kidney are crucial for the maintenance of
phosphate levels.

6. Regulation of phosphate metabolism - where it occurs
Basically, regulation of body's phosphate level can occur at different levels:
-at the site of entry (absorption): it is accomplished by modulating the number
of NaPi2b channels. this mechanism is under regulation by calcitriol (1,25)-OH
vitamin D3 (or 1,25-D3).
-at different sites of use inside the body. Organs and tissue will 'fish'
phosphates in the bloodstream to fulfill their needs. Bone is a heavy consumer
of phosphates both for maintenance of the structure and for the activity of their
cells. Parathyroid is a special organ sensitive to the calcium (and phosphorus)
levels. When calcium rises in the blood, parathyroid will release the hormone
PTH.
-at the site of elimination. In the kidneys, phosphate are first filtered out of the
system (filtration). Then, a specialized mechanism called reabsorption occurs:
phosphates are again filtered out of the urine and reinjected into the
bloodstream. this is a major source of regulation of the blood phopshate.
NaPi2a channels of the proximal tubules are doing this job. Napi2a is under the
regulation of calcitriol (1,25-D3) and of FGF23.

7. the main regulators of phosphate are 1,25-D3, PTH and FGF23. FGF23 is synthesized by
bone cells called Osteocytes and released in the bloodstream. It serves as a signal that is
understood only by kidney cells (those posessing the appropriate receptor called
FGFR1-Klotho) . By an unknown mechanism, FGF 23 is inhibited by PHEX. We will see
more of the regulatory mechanism in the next section below.

8. Regulation of phosphate metabolism - how it occurs?

9. We will look at little bit a the interplay between the different actors of the
phosphate regulation. In the figure , red arrows means a positive effect (
upregulation, stimulation) and obviously blue arrows means a downregulation
(inhibition).
Starting from bone cells: the protein PHEX (mutated in XLH) exerts a
dowregulation on the synthesis of FGF23. So, the level of FGF23 in the blood is
moderate. FGF23 is sensed by kidney cells posessing the co-receptor
for FGF23. This triggers a signal resulting in downregualtion of 1,25-D3 and
NaPi2a. 1,25-D3 is stimulating intestinal absorption of phosphate (via NaPi2b)
and NaPi2a is responsible for the re-aborption of phosphate in the kidney. But
since the FGF23 level is moderate, so is the phosphate wasting signal and
hence, phosphate levels stay balanced.
In XLH, PHEX is mutated and cannot exert its influence on FGF23. As a
consequence, there is an overproduction of FGF23. So, the phosphate wasting
signal is enhanced and this results in the main symptoms of
Hypophosphatemia.

10. Vitamin D Metabolism

11. Phosphate is the major stimulus for 1αhydroxylase.
Reduces 1 α hydroxylation of 25 hydroxy vitamin D
leads to low concentration of 1,25-di hydroxy vitamin D

12. Hypophosphatemic Rickets
• X-linked dominant / Autosomal dominant
• Males affected more than females
• Commonest inherited form of Rickets
• Prevalence 1: 25000
• Phosphate wasting by renal tubules leads to:
– Low serum phosphate
– Normal calcium
• In-appropriate low or normal 1,25-di hydroxy
vitamin D
• Severe rickets and short stature by 1-2 years

13. Which Genes are responsible of HR and I am going to explore them?
1. PHEX: Phosphate regulating Endopeptidase Homolog, X-linked is located on
Xp22.2-p22.1 and it has 22 exons, total annotated spliced Exon length 2861
bp. Mutations in PHEX lead to increased serum levels of FGF23.
2. FGF23: Fibroblast Growth Factor 23 is located on 12p13.3 and it has 3
exons, total annotated spliced exon length 3018 bp. FGF23 causes
downregulation of the renal 1 α hydroxylase enzyme and upregulation of
the 24 hydroxylase enzyme leading to impaired 1,25(OH)2 vitamin D
synthesis and increased degradation. This dual defect in phosphate
metabolism leads to poor bone mineralization and fractures.
3. DMP1: Dentin Matrix Acidic Phosphoprotein 1 is located on 4q21 and it has 6
exons, total annotated spliced Exon length 2687 bp. DMP1 is an
extracellular matrix protein and a member of the small integrin binding
ligand N-linked glycoprotein family. Mutations in the gene are known to
cause autosomal recessive hypophosphatemia, a disease that
manifests as rickets and osteomalacia.

14. Using a DNA extraction kit (Qiagen DNA
Micro, Qiagen, Germany). All 22 exons and their
exon-intron boundaries of the PHEX gene and 3
exons of the FGF23 gene and their corresponding
intron-exon boundaries and 6 exones of DMP1 and
their exon-intron boundaries will be amplified by
PCR and direct sequencing will be carried out to
find the mutations.

15. After sequencing mutations listed in the database include
missense and nonsense mutations, deletions, insertions,
frameshift, and splice site mutations as well as polymor-
phisms. The ethnic distribution of the mutations is very
widespread and includes German, Belgian, Swiss, Spanish,
Italian, French, French-Scottish, Balkan, Polish, Saudi
Arabian, Southeast Asian, African-American, Indian, and
Finnish populations. Only a few mutations have been
reported in Chinese.

16. 1. Xia W, Meng X, Jiang Y, Li M, Xing X, Pang L, Wang O, Pei Y, Yu Ly, Sun Y, Hu
Y, Zhou X. Three novel mutations of the PHEX gene in three Chinese families with
X-linked dominant hypophosphatemic rickets. 2007;81:415-420
2. XLH Research 2012 Phosphate Metabolism (http://www.xlhresearch.net)
3. Harper's illustrated Biochemistry -28th Edition (2000)