1. A Diagnostic Testing Service for Hypophosphatemic Rickets
M. Sloman, K. Thomas, C. Tysoe, S. Ellard
Department of Molecular Genetics

2. Case Study - CB family Diagnosis = Hypophosphatemic Rickets
Rickets at 10 months
I:2
II:2
II:3
Diagnosis = Hypophosphatemic Rickets
III:1
III:2
23 yrs
18 yrs
168cm
163cm
Rickets in early childhood
Dental abscesses
0.74mmol/L
0.76mmol/L
69 mmol/24hr
75 mmol/24hr
Serum Phosphate – Normal mmol/L
Urine Phosphate – Normal mmol/24hr
I first want to introduce you to the CB family referred in 2004 by a local clinical geneticist. The family first presented with this male who when starting to walk at 10 months had bow legs and a diagnosis of rickets was suggested. Rickets is a disease in which bones do not harden sufficiently and become malformed. Features of rickets appear when the child begins to bare weight on their legs causing the legs to bow. His brother also had rickets from an early age and both affected males complained of varying degrees of pain in their legs (which can sometimes be traced to pseudo-fractures). Both males had dental problems with recurrent abscesses and their milk teeth died off very early. Analysis of urine and serum phosphate levels revealed low serum phosphate (known as hypophosphatemia) and high levels of excreted phosphate in the urine. Renal phosphate re-absorption studies revealed a reduced level in both males.
Both males are now 23 and 28 years respectively and their rickets has been treated successfully by surgery and oral supplementation by vitamin D. However both males have been left with below average height.
The clinical diagnosis given to these males was hypophosphatemic rickets.
74%
68%
Renal Phosphate Reabsorption – Normal 82-95%

3. Hypophosphatemic Rickets
Group of disorders associated with childhood rickets, short stature, poor dental development and chronic hypophosphatemia  Defect in renal tubular reabsorption
Most common form X linked dominant
Incidence 1:
X Linked Dominant
Autosomal Dominant
Autosomal Recessive
Hypophosphatemic rickets is a group of disorders associated with chronic hypophosphatemia (low serum phosphate level) which is the result of defects in renal tubular reabsorption (often called renal phosphate wasting). Individuals excrete high levels of phosphate in their urine, known as phosphaturia.
Rare Autosomal dominant and autosomal recessive forms have been reported. The most common form of hypophosphatemic rickets is inherited in an X linked dominant manner (XLHR) with an incidence of 1 in 20,000 births.

4. Clinical Features Childhood rickets Short stature (<50th centile)
Array of bony beads on ribcage
Poor dental development
Varying degrees of bone pain
Osteomalacia
Joint mobility problems
1/3 have hearing problems
Hypophosphatemic rickets is characterised by childhood rickets. Rickets is a disease in which bones do not harden sufficiently and become malformed. Features of rickets appear when the child begins to bare weight on their legs causing the legs to bow. Children with this type of rickets have growth retardation and as adults have short stature ranging from profound to within the normal range around the 50th centile for height. Children with hypophosphatemic rickets have a characteristic array of bony beads on their ribcage, as shown here. Poor dental development is also a feature of the disease with teeth being slow to appear and once erupted dental abscesses are a common problem. Some individuals complain of varying degrees of pain in their legs which can sometimes be traced to pseudo-fractures. These symptoms are often referred to as osteomalacia in adults and literally means softening of the bone due to defective bone mineralisation. Adults have joint mobility problems, particularly with the knees, which can be intermittent or chronic. Joint problems may also be due osteoarthritis which develops due in large part to bone misalignment as a result of childhood rickets or adult osteomalacia. Hearing problems may be a symptom associated with hypophosphatemic rickets. The frequency varies but it is estimated that one third of patients have some hearing difficulties.
The severity of the disease phenotype varies considerably and affected individuals from the same family may have markedly different phenotypes. Some individuals may only have minimal symptoms.
Hypophosphatemic rickets can be treated easily with phosphate supplements and vitamin D preparations. The earlier treatment is initiated to better the outcome, with vast improvements seen in skeletal and dental defects. Stature however remains below the average height.
 Phenotype variable
 Early treatment significantly improves outcome

5. Phosphate Homeostasis
Pi and Ca2+ storage
Pi and Ca2+ absorption
Hypophosphatemia
Pi reabsorption via NPT2
Mobilisation
In order to explain the pathogenic mechanism of hypophosphatemic rickets I must first run through normal phosphate homeostasis. The serum phosphate level is maintained within a narrow range through a complex interplay between intestinal absorption, renal tubular reabsorption via the type ll sodium-dependent phosphate cotransporter and exchange with bone storage pools.
Vitamin D plays an important role in the regulation of body calcium and phosphate levels. 25-hydroxyvitamin D (precursor of Vit D) is hydroxylated in the proximal convoluted tubules of the kidneys to produce the biologically active form of vitamin D, 1,25-dihydroxyvitamin D also called calcitriol. The synthesis of calcitriol is upregulated in hypophosphatemia. The increased serum calcitriol stimulates the absorption of calcium and phosphate from the intestine, enhances mobilisation of calcium and phosphate from bone and increases renal phosphate reabsorption by increasing the activity and number of the brush border membrane NPT2, thus returning serum phosphate levels back to normal.
25-hydroxyvitamin D → 1,25-dihydroxyvitamin D (Calcitriol)
NPT2 = Type ll sodium-dependent phosphate cotransporter

6. Phosphate Homeostasis
Pi and Ca2+ storage
FGF23 = Fibroblast Growth Factor 23
FGF23
Pi and Ca2+ absorption
FGF23
PHEX
DMP1
DMP1 = Dentin Matrix Protein 1
Pi reabsorption via NPT2
FGF23
In addition to Calcitriol, a circulating serum factor (peptide hormone expressed from bone) called Fibroblast growth factor 23 (FGF23) is responsible for regulating serum phosphate levels. FGF23 has the opposite effect to calcitriol in that it inhibits the expression of renal NPT2 and reduces the number transported to the proximal tubule membrane. FGF23 also suppresses the expression of renal 1-hydroxylase the enzyme responsible for the conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D or Calcitriol. The consequence is reduced level of serum calcitriol and loss of its phosphate preserving role.
The level of serum FGF23 is determined by the rate of its cleavage. The PHEX endopepetidase is expressed primarily in bone and by an indirect mechanism promotes proteolytical cleavage of FGF23. Once cleaved FGF23 is inactivated.
Another protein expressed in bone also has an inhibitory effect on FGF23 action. The DMP1 (Dentin matrix protein 1) plays a crucial role in osteocyte maturation in bone and teeth formation. The exact mechanism by which this protein influences FGF23 cleavage is unknown.
25-hydroxyvitamin D → 1,25-dihydroxyvitamin D (Calcitriol)
FGF23

7. Pathogenesis of Hypophosphatemic Rickets
Mutations at cleavage site
DMP1 ?
FGF23
FGF23
PHEX
FGF23
PHEX
Loss of function mutations
FGF23
There are currently three known mechanisms in the pathogenesis of hypophosphatemic rickets. The first by loss of function mutations in PHEX leads to the loss of its promotion of FGF23 cleavage, Loss of function mutations in DMP1 leads to the loss of its as yet unknown mechanism of stimulating FGF23 cleavage and gain of function mutations occurring within the FGF23 cleavage site which prevents its cleavage. All three mechanisms lead to the increase in intact FGF23 level in the serum and as described previously this leads to hypophosphatemia (by decreasing renal phosphate reabsorption and reducing renal Calcitriol production) which in turn leads to hypophosphatemic rickets.
Hypophosphatemia
Hypophosphatemic Rickets

8. Hypophosphatemic Rickets
DMP1
Autosomal Recessive
FGF23
Autosomal Dominant
PHEX
X Linked Dominant
Activating missense mutations around the cleavage site of FGF23 cause the autosomal dominant form of hypophosphatemic rickets. Loss of function mutations in the gene encoding DMP1 cause the autosomal recessive form and loss of function mutations in PHEX cause the most common X linked dominant form.

9. Autosomal Dominant Hypophosphatemic Rickets
Peptide hormone binds renal FGFR2/FGFR4
FGF23
12p13 1 2 3
3 exons
174 P R R H T R S A E 182 6%
R176Q
R176W
R179Q
As I just mentioned the autosomal dominant form of hypophosphatemic rickets is associated with mutations in the Fibroblast growth factor 23 (FGF23) gene. This gene encodes a circulating peptide hormone (found in the serum) which acts via binding to the FGFR2 and FGFR4 receptors both of which are expressed in the kidney. The gene is located on 12q13 and the gene is small with only 3 exons encoding a peptide with 251 amino acids. The cleavage site is located in exon 3 and a close up of the amino acid sequence reveals a 4 amino acid consensus proteolytic cleavage domain flanked by two arginine residues (RXXR) at codons 176 and 179. Cleavage occurs between the arginine residue at codon 179 and the serine at codon 180. The mutations reported in FGF23 are missense mutations affecting the two arginine residues of the cleavage site and block cleavage of FGF23, enhancing/prolonging its action.
In the Exeter laboratory we have sequenced the gene in a total of 18 probands and identified a mutation in 1 proband, giving a mutation detection rate of 6%. The mutation identified affects the arginine residue at codon 179 and was identified in a 4 year old male with extensive family history of hypophosphatemic rickets with an affected father (demonstrating male to male transmission and ruling out the X linked form).

10. Autosomal Recessive Hypophosphatemic Rickets
DMP1
Non-collagenous matrix protein
4q21 1 2 6 3 4 5
6 exons
Lorenz-Depiereux et al 2006 Nat Genet 38:1248
Feng et al 2006 Nat Genet 38:1310
12%
M1V (c.1A>G)
c.55-1G>C
c.362delC
c.1484_1490del
Mutations in the DMP1 gene were shown to cause autosomal recessive hypophosphatemic rickets in The gene encodes a non-collagenous matrix protein which is essential in osteocyte maturation in bone and tooth formation. Its role however in FGF23 cleavage is unknown. The gene is located on 4q21 and has 6 exons with exon 1 being non-coding. The gene encodes a peptide with 514 amino acids. To date 4 different mutations have been identified, 2 frameshift, 1 splicing and 1 affecting the initiation codon, all of which lead to the loss of DMP1 function. Mutation analysis of 42 probands in two separate studies identified mutations in 5 affected individuals giving a mutation detection rate of 12%.

11. X Linked Dominant Hypophosphatemic Rickets
PHEX
Endopeptidase
Xp22.1
22 small exons (17 less than 130bp)
193 mutations reported (
The PHEX endopeptidase is encoded by the PHEX gene. The gene is located on Xp22.1 and contain 22 small exons 17 of which are less than 130bp in size. The intron however are large. To date 193 different mutations have been reported and are available through the PHEX mutation database. The mutation spectrum is wide including gross deletions and mutations are spread across the coding region with no mutation hotspots. All mutations lead to loss of function and are the cause the X linked form of hypophosphatemic rickets

12. PHEX Analysis by Sequencing
Set up in 2002
All coding exons screened by sequencing
71%
Screening for the PHEX gene was set up in the Exeter laboratory in 2002 and all coding exons are screened using sequence analysis. Since 2002 we have tested 113 probands and identified a mutation in 80 of them, giving a mutation detection rate of 71%.

13. PHEX Deletions Identified in Males
3 Deletions picked up in affected males (3/38 = 8%)
On analysis 3 affected males were shown to have partial gene deletions from a total of 33 males analysed by sequencing. These were evident from the absent of PCR products for certain exons on agarose electrophoresis in these hemizygous males. One male had a single exon deletion of 16 and another of exon 13 (gel image not shown). The third male had a deletion of exons 17 through to 22. Partial gene deletions have been reported in the literature and they are shown here in blue. One study using PCR and southern blotting identified 7 affected males with deletions out of a group of 33 affected individuals (?sex) with mutations in the PHEX gene, which shows that deletions may represent up to 21% of mutations in the PHEX gene.
Francis et al 1997 Genome Res 7: 573
21% (7/33) mutations were deletions

14. PHEX MLPA Validation Confirmed deletions identified in males
PHEX Kit - P223 MRC Holland
Deletion exon 17-22
Deletion exon 16
Deletion exon 13
Following the introduction of an MLPA kit for PHEX by MRC Holland in 2007 we confirmed the three deletions in the affected males. The MLPA data is displayed here in genemarker and when using males as controls. The Y axis represents the peak ratio or dosage quotient and the X axis the product size. The green points represent probes within the PHEX gene and blue the control probes. The lines represent the standard deviation within which DQ are classed at normal dosage. Note peak ration for deletions in males in zero.

15. Dosage Analysis of PHEX
Heterozygous deletions identified in 9/17 (53%) females tested
Whole gene deletion
Deletion exon 13-14
Deletion of exon 16
17 affected female patients in whom a mutation was not identified by sequencing were analysed using MLPA (13 retrospective and 4 new referrals). Dosage analysis identified 9 females with PHEX deletions including single exon deletions, partial gene deletions such as exon and a whole gene deletion.
It is worth noting the apparent clustering of deletions and due to this MRC Holland put 2 probes in exons 1, 3, 5, 11, 12, 15.

16. Dosage Analysis of PHEX
Duplications identified in 2 females
Duplication exon 13-14
Duplication exon 13-20
In addition to the nine females with deletions 2 affected females were found to have large duplications. One duplication was of 2 exons 13 to 14 and a second duplication covering exons 13 to 20. Duplications have not been previously reported in the literature. This finding has implications for screening males as duplications unlike gross deletions will not be identified by a PCR/sequencing strategy.
Duplications not previously reported in the literature
Implications for males

17. PHEX Screening Strategy
Screening for all affected individuals = Sequencing and MLPA
81%
71%
Hence our screening strategy for all affected individuals both males and females is sequencing followed by MLPA. This strategy has now been implemented into the diagnostic service and has increased the PHEX mutation detection rate from 71% to 81%.

18. Case Study - CB family 18 yrs Screened for mutations in PHEX
III:2
II:2
II:3
18 yrs
Screened for mutations in PHEX
Now returning to the CB family. The pedigree resembles autosomal recessive inheritance. However the family were referred in 2004 before the DMP1 gene for autosomal recessive hypophosphatemic rickets was known. Instead KCB was screened for mutations in the PHEX gene the most common cause of hypophosphatemic rickets.
III:1
23 yrs

19. Case Study - CB family Novel missense mutation exon 5
p.Pro168Leu (c.503C>T)
I:2
Normal stature N
N/N
Pathogenic?
Highly conserved
Different side chains
FGF23 negative
II:2
II:3
p.Pro168Leu/N
De novo in II:2?
II:2 mosaic?
II:2 Buccal
II:2 Blood
163cm
86%
(82-95%)
0.85
( mmol/L)
Now returning to the CB family. Following PHEX sequencing in KCB a novel missense mutation P168L was identified in exon 5. This mutation was also identified in the affected brother JCB. Testing for the mother HCB was carried out and she was found to carry the mutation. HCB however was known to have normal tubular phosphate reabsorption. Samples from uncle PC and grandmother WC were also available for testing and both were shown not to carry the P168L mutation. No samples were available from the deceased grandfather but he was known to be of normal stature. The finding of P168L in the unaffected mother did not fit with X linked dominant inheritance and as this mutation has not been reported previously its pathogenicity was uncertain. The Proline at codon 168 is highly conserved and proline and leucine have very different side chains. Analysis using bioinformatic tools such as Sift and polyphen indicated that the mutation is probably damaging. So there is some evidence for P168L being pathogenic. If the P168L was not pathogenic then the hypophosphatemic rickets seen in KCB and JCB could be caused by mutations in other genes. We sequenced FGF23 in KCB, as this was the only other HR gene set up for analysis in lab, and did not identify a mutation.
So if the P168L mutation is pathogenic then it is possible that it is de novo in HCB and that she is a mosaic. However analysis of DNA from different tissue origins namely Blood lymphocytes and buccal cells showed the P168L mutation and at the same mutation load. This however does not rule out mosaicism in other tissues e.g. Bone where PHEX is highly expressed. Another alternative is that HCB inherited the mutation from her father whose exact status is unknown but is known to be of normal stature. To resolve this we carried out linkage analysis on the family to determine the grandparental origin of P168L.
 Linkage analysis
III:1
III:2
p.Pro168Leu
p.Pro168Leu
23 yrs
18 yrs

20. Grandfather and male siblings normal stature
N/N
Grandpaternal origin
246
215
353
212
287
253
266 85
319
159
155
202
124
114
252
203
353
218
283
243
266 87
325
161
155
204
124
114 83
242
203
355
216
281
253
282 83
321
161
147
210
126
110 75
Grandfather and male siblings normal stature
 De Novo
Skewed X inactivation?
II:2
II:3
Normal X inactivation ratio
p.Pro168Leu/N N
246
215
353
212
287
253
266 85
319
159
155
202
124
114
252
203
353
218
283
243
266 87
325
161
155
204
124
114 83
252
203
353
218
283
243
266 87
325
161
155
204
124
110 75
Serum FGF23 Level
109U/ml
Normal <100U/ml
p.Pro168Leu causes loss of PHEX function
III:1
III:2
We used 15 microsatellite markers spread across the X chromosome and showed that all individuals with the P168L mutation inherited the A haplotype which is of grandpaternal origin. From this it is not possible to say whether the mutation is de novo for certain but since the grandfather and his male siblings are all of normal stature it implies that P168L may be de novo.
A further possible explanation for that lack of phenotype in HCB is skewed X inactivation where a higher proportion of the X chromosome carrying P168L is inactivated. However testing at the Salisbury laboratory revealed a normal x inactivation ratio.
Another investigation carried out on HCB was measurement of serum FGF23 level, where PHEX is responsible for promoting the cleavage and inactivation of FGF23. This was carried out in a biochemistry lab in Liverpool using an ELISA method and revealed a level of 109 in HCB where the normal range is <100U/ml. This suggested that P168L cause loss of PHEX function and is consistemat with XLD hypophospahetmic rickets. It was concluded therefore that II:2 was mildly affected and that P168L is pathogenic.
p.Pro168Leu
p.Pro168Leu
II:2 mildly affected
DXS1060
252
215
353
212
287
243
266 87
325
161
155
204
124
114 83
252
215
353
212
287
253
266 85
325
161
155
202
124
114
DXS987
DXS1226
DXS1202
DXS1214
= Location of PHEX gene
Key:
= Haplotype C
= Haplotype B
= Haplotype A
DXS1068
DXS993
DXS1055
DXS991
DXS986
DXS990
DXS1106
DXS1001
DXS1047
DXS1227

21. Summary Hypophosphatemic rickets inherited in AD, AR and XLD manner
Childhood rickets, short stature, poor dental development and hypophosphatemia
Genetic diagnosis enables early treatment and significantly improves outcome
Screening available for FGF23 and PHEX genes  Mutation detection rate of 6% and 81% respectively
Gene dossier submitted for DMP1 in February 2008

22. Acknowledgements Dr Julia Rankin Dr Carole Brewer
Exeter Laboratory team
I would just like to thank Katie Thomas for all her work on the PHEX MLPA, Dr Julia Rankin for referring the CB family and the staff at the Exeter laboratory particularly …. Who have carried out screening of all the patients mentioned in this presentation.