23/02/2014

Dr Geeta Hampson
Consultant Chemical Pathologist
GSTT

Vitamin D Metabolism
The “Secret” Life of Vitamin D

(Animals, Supplements)
(Plants)

(Calcitriol: Active form of Vit D)

Initial investigation of hypercalcaemia

History/examination
Exclude drugs : thiazides,
lithium
Albumin-corrected calcium> 2.65
Malignancy
mmol/L
Normal renal function
Measure PTH 1. Humoral hypercalcaemia of malignancy.
concentration
PTH > 65 ng/L PTH <65 ng/L
2. Ectopic production of 1,25(OH)2 D3 (lymphoma).
Primary
Malignancy Urine Ca cl /Cr cl <0.01
hyperparathyroidism 3. Lytic bone metastases.
(not excluded) ? FBHH
Tertiary (rare)
Non-parathyroid causes :
malignancy, myeloma,
Dual pathology
vitamin D excess,
possible
thyrotoxicosis, sarcoid

PTH-rp > 2 pmol/L

Malignancy very likely

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Circulating PTHrP Concentrations in HHM: Parathyroid Hormone-related Protein

Two-Site IRMA (1986)

1 13 84
PTH
(Mr 9600)

1 13 141
PTHrP
(Mr 16,000)

1 2 3 4 5 6 7 8 9 10 11 12 13......
PTH Ala Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys .....

PTHrP Ala Val Ser Glu His Gln Leu Leu HisAsp Lys Gly Lys .....

Burtis WJ…Stewart, AF. N Engl J Med 322:1106-1112, 1990

Clinical Biochemical Features of HHM: 1980

PTH-PTHrP Signaling via the PTH-R

PTH PTHrP TmP/

NcAMP
GFR
Out
Bone
Kidney
In
Ca++ 1,25
Excretion (OH)2D

PKA, cAMP PKC, Cai++

Stewart, Broadus et al. N Engl J Med 303:1377-1383, 1980

Primary hyperparathyroidism Familial Hypocalciuric Hypercalcaemia

Relatively common, 1 : 500-1 : 1000 Autosomal dominant.
• Moderate Hypercalcaemia.
Post-menopausal women • Relative hypocalciuria, Ratio of Calcium clearance to creatinine clearance
80% : benign solitary adenoma low (Less than 0.01).
• PTH - Normal. (inappropriate)
15-20% : parathyroid hyperplasia
FBBH Primary HPT
0.5% : parathyroid carcinoma
Age (years) <40 >50
Clonal origin of parathyroid adenomas
Sex Equal men and Mainly women
women
Changing clinical presentation of primary
Symptoms Unrelated to Related to calcium
hyperparathyroidism
calcium
1930-1970 1970-2000
Albumin 2.55-3.5 2.55-4.5
Nephrolithiasis 51-57% 17-37% Corrected calcium
Hypercalciuria 36% 40% PTH <65 ng/L >65 ng/L
Overt skeletal 10-23% 1.4-14% Magnesium 0.78-1.18, 0.34-1.03
disease 1,25 (OH)2 vit D 54-134 pmol/L Often raised
Aymptomatic 0.6-18% 22-80% Ca cl/Cr cl <0.01 >0.015

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Familial Hypocalciuric Hypercalcaemia Clinical approach in the investigation of hypocalcaemia

Genetics Hypocalcaemia
• autosomal dominant, 100% penetrance. Serum adjusted calcium <2.2 mmol/L
• most cases ® inactivating mutations in the calcium - sensing or ionised calcium < 1.1 mmol/L
receptor. in glandular ‘setpoint’ for calcium suppression of PTH secretion.
• Loss of receptor function Clinical assessment : for risk factors for vitamin D deficiency and for common causes
• Over 130 different mutations identified of hypocalcaemia (e.g CKD, post-surgical hypoparathyroidism or drugs

Other biochemical tests

First line tests : Phosphate, Magnesium , Creatinine/GFR, PTH

Amylase, Creatinine kinase, 25 (OH)vitamin D

Reduced phosphate Raised phosphate + Raised phosphate +

+ raised PTH reduced PTH raised PTH

• Homozygotes Þ neonatal severe primary hyperparathyroidism

• CASR mutations detected in 65% of FHH Vitamin D deficiency Hypomagnesaemia CKD
Acute pancreatitis Hypoparathyroidism Massive tumour lysis
• FHH3 (gene encoding adaptor protein 2 sigma subunit which regulate levels of Drugs (bisphosphonates) Drugs (cinacalcet) Early rhabdomyolysis
CASR cell surface expression)
• FHH2 (loss of function mutation in Gα 11, involved in CASR signalling)

Causes of hypoparathyroidism
Inadequate PTH Post-surgical, drugs Gene
Regulation of phosphate homeostasis
secretion (Cinacalcet) defect Parathyroid Phosphorus absorption in the gut Organification
Infiltrative diseases (mostly passive) into nucleic
(haemochromatosis, wilson’s acids, etc.
disease, metastatic cancer)
Neck irradiation
Pi Bone mineral
Magnesium disorders
1,25 VitD
Genetic causes PTH PTH
autosomal GCMB
FGF23
x-linked SOX3
NHERF1
Autoimmune disorders : AIRE
isolated, auto-immune poly
glandular syndrome
Phosphatonin
1,25 VitD
Genetic syndromes : Bone
Di George syndrome TBX1
Proximal
Activating CASR mutation, CASR Tubular Cell
HDR syndrome GATA3
Resistance to Pseudohypoparathyroidism GNAS
Filtered
PTH action (type 1a, 1b, 2) NHERF1 NPT2a NPT2c

Fibroblast Growth Factor-23 (FGF-23) Molecular mechanisms of phosphate disorders

NaPi-IIa NaPi-IIc

ADHR ¯ 1,25 (OH)2 vit D

Proximal
tubular cell
FGF23-R?

Klotho
phosphatonin

FTC-2
FGF23 FGF23m

PHEX Inactive FGF-23 High C-term.

sFRP4 low intact FGF23
MEPE Normal
TIO osteoblast/osteocyte mutant
GALNT3 FTC-1
DMP-1

FGF7
Primer on metabolic bone disease; ASBMR publication

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ADHR is caused by FGF23 mutations

Autosomal Dominant Hypophosphatemic Rickets (ADHR)

01 MLGARLRLWVCALCSVCSMSVLRAYPNASP ~15 kDa ~8 kDa

• Clinical findings: 31 LLGSSWGGLI HLYTATARNSYHLQI HKNGH 25 176-9 251
- Variable age of onset 61 VDGAPHQTI YSALMI RSEDAGFVVI TGVMS
91 RRYLCMDFRGNIF GSHYFDPENCRFQHQTL
- Lower extremities deformities, if onset before puberty 121 ENGYDVYHSPQYHFLVSLGRAKRAFLPGMN RXTR:
- Rickets/osteomalacia 151 PPPYSQFLSRRNEI PLIH FNTPI PRRHTRS
181 AEDDSERDPLNVLKPRARMTPAPASCSQEL
- furin-like proprotein
convertase
211 PSAEDNSPMASDPLGVVRGGRVNTHAGGTG
• Biochemical findings: 241 PEGCRPFAKFI - O-linked sugar
- normal serum calcium
- low serum phosphorous (variable)
- increased renal phosphate excretion (variable)
- normal 25-hydroxy vitamin D
- “inappropriately” normal or low 1,25(OH)2D
- normal PTH White, et al.; Nat. Genet. 2000
WT Mut.
Econs et al.: JCEM, 1997

Tumour-induced osteomalacia
TIO disorder of renal phosphate wasting associated with a benign
• Acquired
FGF23 is overexpressed in TIO tumors
tumour
• Osteomalacia is seen on bone biopsies
Tumor 1 2 3 4 5 ctr.
3 kb

- 32 kDa
1.3 kb

• 80% : mixed connective tissue tumours, 3 other categories :

osteoblastoma-like tumours, nonossifying and ossifying fibroma-like
tumours Western Analysis
Northern probe: 30 min. exposure
• Tumours secrete one or more substances that interfere with renal cDNA encoding
phosphate handling and vitamin D metabolism White et al.: JCEM, 2001
human FGF- 23

FGF23 concentrations in TIO

Elevated FGF23 levels in XLH

8000 ADHR TIO 8000

6000 FGF23act FGF23 6000 WT Hyp

_________________________
4000 4000 FGF23 123±11 2193±159
(pg/ml)
2000 2000
FGF-23 (RU/ml)

FGF-23 (RU/ml)

Liu et al.: JASN, 2005

FGF23-R?

700 Proximal 700

Tubular Cell
600 600
500 500
400 400
300 NaPi-IIa NaPi-IIc 300
200 200
100 Adult/pediatric normal range 100 Adult/pediatric normal range
0 0
before after before after XLH
tumor removal
Jonsson et al.: NEJM, 2003 tumor removal
Jonsson et al.: NEJM, 2003

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Fibrous Dysplasia :
Familial Tumoural Calcinosis (FTC)
activating mutations of the GNAS gene FTC is an autosomal recessive disorder characterised by
• hyperphosphataemia,
• high 1,25 (OH)2 vit D
• Monostotic/polyostotic • and severe ectopic calcifications
• Extra-skeletal manifestations (cutaneous hyper pigmentation and endocrinopathies • Genes implicated : FGF-23,
(MAS) • GALNT3 Mutational analysis of FGF-23 :
• Renal tubulopathy (renal phosphate wasting) • Loss of function mutation Histidine to Glutamine change
in KLOTHO

Masi et al
J Bone Joint Surg Am 2009;
91: 1190-1198

Fibroblast Growth Factor 23 (1), 1,25-dihydroxyvitamin D (2), Parathyroid

Hormone (3) and Phosphate (4) levels (y axis) according to Glomerular
Second v/s third-generation PTH assays
Filtration Rate (x axis).

• N-form of PTH is detectable by the

3rd generation assay but poorpy
reactive in the 2nd generation assay.
• ? Biological active
• Seen in severe hyperparathyroidism
and parathyroid carcinoma

Second v/s third-generation PTH assays in ESRD and CKD FGF-23 in Chronic Kidney Disease (CKD)
Bland-Altmann plots of the 2 PTH assays in ESRD and CKD

ESRD
P < 0.001

CKD P < 0.05

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FGF-23 cleavage site and ELISA techniques used to Fibroblast Growth Factor-23 : ? Direct function on vessel wall
detect intact and C-terminal fragments

Inaba et al 2006 Osteoporos Int 17:1506–1513

Gutierrez et al 2008, New Engl J Med 359; 6: 584-

592

Aortic arch calcification score in haemodialysis patients

Tamei et al 2011 J Atheroscler Thromb 18, 217-223