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

A Diagnostic Testing Service for Hypophosphatemic Rickets

M. Sloman, K. Thomas, C. Tysoe, S. Ellard

Department of Molecular Genetics

Case Study - CB family

III:1 III:2

I:2

II:2 II:3

Diagnosis = Hypophosphatemic Rickets

Rickets at 10 months Rickets in early childhoodDental abscesses Dental abscesses 0.74mmol/L 0.76mmol/L69 mmol/24hr 75 mmol/24hr

Serum Phosphate – Normal 0.8-1.4mmol/LUrine Phosphate – Normal 10-32 mmol/24hr

74% 68%

Renal Phosphate Reabsorption – Normal 82-95%

23 yrs 18 yrs

168cm 163cm

Hypophosphatemic Rickets

Autosomal Dominant

Autosomal Recessive

Most common form X linked dominant

Incidence 1: 20 000

X Linked Dominant

Group of disorders associated with childhood rickets, short stature, poor dental development and chronic hypophosphatemia Defect in renal tubular reabsorption

Phosphate Homeostasis

Pi and Ca2+ absorption

Pi and Ca2+ storage

Pi reabsorption via NPT2

NPT2 = Type ll sodium-dependent phosphate cotransporter

25-hydroxyvitamin D → 1,25-dihydroxyvitamin D (Calcitriol)

Hypophosphatemia

Mobilisation

Phosphate Homeostasis

Pi and Ca2+ absorption

Pi and Ca2+ storage

Pi reabsorption via NPT2

25-hydroxyvitamin D → 1,25-dihydroxyvitamin D (Calcitriol)

PHEX

FGF23

FGF23 = Fibroblast Growth Factor 23

FGF23

FGF23FGF23

FGF23FGF23

FGF23

FGF23

FGF23

DMP1

DMP1 = Dentin Matrix Protein 1

Pathogenesis of Hypophosphatemic Rickets

FGF23

PHEX

FGF23

FGF23

PHEX

FGF23

Hypophosphatemia

Hypophosphatemic Rickets

Loss of function mutations

Mutations at cleavage site

DMP1

?FGF23

FGF23 FGF23

FGF23FGF23

FGF23 FGF23

FGF23FGF23

Hypophosphatemic Rickets

FGF23

Autosomal Dominant

PHEX

X Linked Dominant

DMP1

Autosomal Recessive

1 2 3

3 exons

Autosomal Dominant Hypophosphatemic Rickets

FGF2312p13 Peptide hormone binds renal FGFR2/FGFR4

R176QR176WR179Q

174 P R R H T R S A E 182

17

10

2

4

6

8

10

12

14

16

18

Mutation Negative Mutation Positive

Nu

mb

er o

f P

rob

and

s

6%

DMP1

Autosomal Recessive Hypophosphatemic Rickets

4q21

1 2 63 4 5

6 exons

Non-collagenous matrix protein

M1V (c.1A>G)

c.55-1G>C c.362delCc.1484_1490del

Lorenz-Depiereux et al 2006 Nat Genet 38:1248

Feng et al 2006 Nat Genet 38:1310

37

50

5

10

15

20

25

30

35

40

Mutation Negative Mutation Positive

Nu

mb

er

of

Pro

ba

nd

s

12%

X Linked Dominant Hypophosphatemic Rickets

PHEX Endopeptidase

193 mutations reported (www.phexdb.mcgill.ca)

22 small exons (17 less than 130bp)Xp22.1

PHEX Analysis by Sequencing Set up in 2002All coding exons screened by sequencing

33

80

0

10

20

30

40

50

60

70

80

90

Mutation Negative Mutation Positive

Nu

mb

er

of

Pro

ba

nd

s 71%

PHEX Deletions Identified in Males

Francis et al 1997 Genome Res 7: 573

21% (7/33) mutations were deletions

3 Deletions picked up in affected males (3/38 = 8%)

PHEX MLPA Validation

Confirmed deletions identified in males

PHEX Kit - P223 MRC Holland

Deletion exon 17-22 Deletion exon 16

Deletion exon 13

Dosage Analysis of PHEX

Heterozygous deletions identified in 9/17 (53%) females tested

Deletion exon 13-14 Whole gene deletion

Dosage Analysis of PHEX

Duplications identified in 2 females

Duplications not previously reported in the literatureImplications for males

Duplication exon 13-20Duplication exon 13-14

PHEX Screening Strategy

Screening for all affected individuals = Sequencing and MLPA

8092

3321

0

20

40

60

80

100

120

Mutation detection rate onSequencing

Mutation detection rate onSequencing and MLPA

Nu

mb

er

of

Pro

ba

nd

s

Mutation Positive Mutation Negative

81%71%

Case Study - CB family

III:1

23 yrs

I:2

III:2

II:2 II:3

18 yrs

Screened for mutations in PHEX

Case Study - CB family

I:2

III:1 III:2

II:2 II:3

23 yrs 18 yrs

Novel missense mutation exon 5

p.Pro168Leu (c.503C>T)

p.Pro168Leu p.Pro168Leu

N

N/N

p.Pro168Leu/N

Normal stature

Pathogenic?

Highly conservedDifferent side chainsFGF23 negative

De novo in II:2?II:2 mosaic?

Linkage analysis

163cm

86% (82-95%)

0.85 (0.8-1.4 mmol/L)

II:2 Buccal II:2 Blood

25221535321228724326687325161

155204

124114

83

25221535321228725326685325161

155202

124114

85

24621535321228725326685319159

155202

124114

85

25220335321828324326687325161

155204

124114

83

25220335321828324326687325161

155204

124110

75

24621535321228725326685319159

155202

124114

85

25220335321828324326687325161

155204

124114

83

24220335521628125328283321161

147210

126110

75

DXS1060DXS987DXS1226DXS1202DXS1214DXS1068

DXS986DXS990

DXS993DXS1055DXS991

DXS1106DXS1001DXS1047DXS1227

p.Pro168Leu p.Pro168Leu

p.Pro168Leu/N N

N/NI:2

III:1 III:2

II:2 II:3

= Location of PHEX gene

Key:

= Haplotype C= Haplotype B= Haplotype A

Grandpaternal origin

Grandfather and male siblings normal stature De Novo

Skewed X inactivation?Normal X inactivation ratio

Serum FGF23 Level

109U/ml

Normal <100U/mlp.Pro168Leu causes loss of PHEX function

II:2 mildly affected

Summary

Hypophosphatemic rickets inherited in AD, AR and XLD manner

Gene dossier submitted for DMP1 in February 2008

Screening available for FGF23 and PHEX genes Mutation detection rate of 6% and 81% respectively

Genetic diagnosis enables early treatment and significantly improves outcome

Childhood rickets, short stature, poor dental development and hypophosphatemia

Acknowledgements

Dr Julia Rankin

Dr Carole Brewer

Exeter Laboratory team