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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
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
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