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Genetics and The Biology of Bone: EFF

Clifford J Rosen

Maine Medical Center Research Institute

Scarborough, Maine 04074

rosenc@mmc.org

Outline• Brief overview of bone biology

• Genetics of osteoporosis– Mouse- humans; humans-mouse

• Skeletal physiology from a mutational perspective

• Clearer insights into remodeling– HBM

– SFRP4

– CatK

– GGPS1

• Clinical Translation

Three Major Take Home Points• Bone is fully integrated with metabolic

homeostasis; hence bone IS NOT distinct from other organs

• Mutations, even rare ones, found by GWAS provide tremendous insights into physiology and pharmacogenetics

• Fuel Homeostasis in the bone marrow niche is an essential part of skeletal remodeling and important in anabolism

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SKELETAL PHYSIOLOGY: Critical Phases

• Skeletal Growth– Prenatal to Adolescence– Modeling and remodeling– Early and Rapid linear growth which then slows before

increasing with adolescence

• Peak Bone Acquisition– Ages 12-18– Gender and compartment specific!!!!– Strong genetic determinants– Phased with linear growth

• Bone Maintenance– Ages 20-50: Remodeling 10% of

the skeleton/yr

• Bone Loss– Gender and compartment specific– Genetic determinants may play a role

1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0

A c tiv eG r o w th

S lo wL o s s

R a p idL o s s

L e s s R a p idL o ss

Bone Remodeling Process

Osteoclasts

Lining Cells

Bone

Lining Cells

MineralizedBone

ResorptionCavities

Osteoblasts

Osteoid

OC OB

H+

FormationResorption20 Days 100 Days

RANK-L(OPGL)

Osteocalcin

BSAP

PICP

IL-1, PTH, IL-6, – E2, IGF-I

M-CSF

120 Days

Collagen

Proteases

Osteocalcin

Matrix

CTxNTx

D-PyrRANK OPG

Bone Remodeling: Coupling Formation toResorption and resorption to formation

IGFsIGF-BPsTGFs

αvβ3

IGF-IIGFBPs

LRP4,5,6

Osteocyte

Sclerostin

-Tgf-β

COOH

Cthrc1, BMP-7,S1P,Wnt10b

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The Bone Marrow Niche- Multiple Cell Types- All Contribute to Homeostasis

SNS

BAT “Beige or Bright”

SNSSNS

Wnt10bIgfbp2

irisin

Cthrc1

Pgc1a

BDNF

adiponectin

unOC

Bone is Connected to MetabolicHomeostasis

Common Circuits:

BoneMuscle

Sarcopenia Osteopenia BAT dystrophy Lipoatrophy

TSH FSH, ACTH

The CNS Regulates Bone Turnover

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Bone Regulates the CNS!!!

SostFGF-23

Signal Integration- Mechanical, chemical, metabolic

Others?

BONE LINING CELL: A FIBROBLAST IN THE BONE MARROW-

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• PBM is a determinant of lifetime BMD

• BMD is the major determinant of future fracture

• Multiple factors contribute to PBM

• Genetic determinants are critical but are modifiable by environmental factors

Genetics60%

Lifestyle

PEAK BONE MASS12-22 years of age HormonesNutrition

10 20 30 40 50 60 70 80 90

A ctiveG row th

S lowL oss

R apidL oss

L ess R ap idL oss

Peak Bone Mass is a Quantitative Trait and is Complex!

• BMD is a Quantitative trait– Other examples: hypertension, music appreciation, height,

weight,• Not simple mendelian inheritance• Multiple genes contribute to the quantitative

phenotype• BMD: distributed in a gaussian manner

• Gene-gene interaction common• Environmental interaction characteristic• Epigenetic influences difficult to distinguish

0 +2.5-2.5

Approaches to Define Genes Related to Peak Bone Mass

• Candidate gene approach– Regular candidates related to bone remodeling

• Twin studies, Pair studies

• Whole genome scanning to detect candidates in large unrelated populations– DeCode- Iceland; China, Genetics Consortium-

GEFOS – 99% of genome is non coding

– Deep sequencing- major advance that provides significant confirmation and accuracy

– Whole exome sequencing readily available for the 1% coding regions.

– Rare variants

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Candidates genes for osteoporosis: bone remodeling

Receptors • Vitamine D Receptor (VDR)• Estrogen receptors• Calcitonin receptor• Calcium sensing receptor• PTH• Androgen• Osteoprotegerin• Glucocorticoids

• Tumor necrosis factor

Bone-associated proteins• Collagen type 1• Osteocalcin • Bone matrix protein

Growth factor and cytokines• Interleukin 6• TGF- Beta• IGF-I• Bone morphogenetic protein 2 • Interleukin-1 receptor antagonist• Tumor necrosis factor alpha

• LRP-5Enzymes • Aromatase• Methylenetetrahydrofolate

reductase

Signaling moleculesIRS-1

Miscellaneous• Apolipoprotein E• Heparin sulfate glycoprotein

Genome Wide Association Studies (GWAS)

• Collect populations (1-1,000, 000)• Measure phenotype

– BMD, other bone mass phenotypes– Bone turnover marker– Fracture

• Define polymorphisms in non-candidate genes; generally non coding

– Millions of SNPs,micro satellites, – Haplotype association mapping HAM– Deep sequencing- second generation-WES

• Perform genotype-phenotype association• Determine statistical association and test in

other cohorts –Meta analysis

GWAS StudiesLimitations Strengths

Many false positives

Some false negatives

Population heterogeneity

Multiple comparisons lead to chance associations

Genes do not assign function- but!!!!!

Getting less EXPENSIVE!!!

• Large cohorts

• Larger cohorts

• Larger and larger cohorts

• Refined statistical and genetic analyses

• Test human genes in mice

• Can now do meta-analyses

• GWAS can define fx

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GEFOS Consortium

133,000 samples44,000 + GWA

Gene Discovery Requires Validation Cohorts

Hsu, PLOS genetics2010

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Rare SNVs and CNVs and Their Importance for Bone

J Med Genet. 2014 Feb;51(2):122-31. doi: 10.1136/jmedgenet-2013-102064. Epub 2013 Dec 16. A genome-wide copy number association study of osteoporotic fractures points to the 6p25.1 locus

J Clin Endocrinol Metab. 2014 Nov;99(11):E2400-11. doi: 10.1210/jc.2014-1584. Epub 2014 Aug 13. Common and rare variants in the exons and regulatory regions of osteoporosis-related genes improve osteoporotic fracture risk prediction.Lee SH1, Kang MI, Ahn SH, Lim KH, Lee GE, Shin ES, Lee JE, Kim BJ, Cho EH, Kim SW, Kim TH, Kim HJ, Yoon KH, Lee WC, Kim GS, Koh JM, Kim SY

How has genetics opened up new aspects of bone biology and new

treatments?

Clinical ObservationTo Basic Lab

To Genetic StudiesTo the Clinic

Back to the lab!!!

Osteoporosis Pseudo Glioma(Matt Warman) Very RARE!Autosomal Recessive

High Bone Mass: Z Score 5.6 !(R. Recker and M Johnson)Autosomal Dominant

Both linked to chromosome 11q13

Human Genetic Studies Link Bone Mass To a Specific Region of the Human Genome

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LDL rec repeats

B propellers

EGF-like repeats

HBM and OPPG turned out to be mutations Low Density Lipoprotein Receptor Related P5

LRP 5

HBM G171V mutation

OPPG Null or SinglePoint mutations

The Wnt RegulatorySystem

Canonical and Non Canonical Signaling

Wnt 5aWnt 3a

Inc bone IncreasedFormation bone resorption

sFRP4

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Shoback D, J Clin Endocrinol Metab, 2007; 92: 747–753

Sclerostin is a potent inhibitor of the Wnt/β-cat pathway

Monoclonal to Sclerostin vs Alendronate and PTH

McClung NEJM 2014

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Romozsozumab Reduces Fractures

The Phenotypic Range of Genotypic Variation inOne Gene!!!

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Another Example of Rare Genetic Defecs: Cathepsin K 

Deficiency  Pycnodysostosis is a genetic disease

associated with cathepsin K deficiency (Gelb,et al.,1996; Schilling et al 2007)

Rare autosomal recessive skeletal dysplasia ~150 cases of pycnodysostosis reported

worldwide Short stature, acrosteolysis of distal

phalanges and large fontanelles High bone mass and increased fragility

associated with high risk of fracture Normal intelligence, sexual development and

lifespan

Cat K null mice have osteopetrotic phenotype, but otherwise healthy

Frontal bossing

Schilling et al 2007

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Normal

Osteoclasts resorb lessOsteoblasts form boneGreater gain in BMD

Odanacatib

Lotinun, Clin Invest. 2013

per 10,000person years treatment

Schilcher et al Acta Orthop 2015;86(1)100-107

Atypical femur fractures: Relative riskAtypical Femoral Fractures

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Going from mouse to humans and back

Rationale for Studying Mice

• 40 Inbred strains– Provide large number of

identical “twins” for linkage studies– Controlled breeding-environment-diet– Relatively Short generation times– In silico data bases are superb

• Mouse genome can be manipulated to test function of particular candidate gene

-Knockout, knock in, temporal and context specific changes

New technology shortens time to generation of mouse models- CRSPR/Cas9

0.5 mm

Total femur vBMD by pQCT in Females-5 Inbred Strains

0.20

0.30

0.40

0.50

0.60

0.70

1 2 4 8 12Beamer et al, Bone, 1996

TotalFemurvBMD(g/cm3)

C3H/HeJ (C3H)

C57BL/6J (B6)

Age (months)

PEAK vBMD

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Skeletal and Marrow Differences in B6 vs C3H

C57

BL

/6J

C3H

/HeJ

Cortical Trabecular Marrow Fat

?

Adipocyte to Bone and Back

PTH

Myf5 dTomato, Adipo GFP, Pdgfrα 50% GFP +

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Morrison et al Cell Stem Cell, 2016

Potential Metabolic Pathways in the Osteoblast

Glycolysis 1mol glu=2ATPGlutamine 1 mol 9 ATPFatty acids 1 mol = 36 ATP

PTH Induces Glycolysis over OxPhos Ex Vivo

0

5

10

15

20

25

EC

AR

(m

pH

/min

)

VE

H

PT

H 1

00n

M

PT

H 1

50n

M

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PTH Treatment Changes BMAT in Men

Summary-• SNVs in particular gene loci contribute

to variation in complex traits ; e.g. obesity, BMD; rare variants may lead to greater contributions to genetics

• New tools can identify enhancers and repressors that regulate downstream genes

• CRISPR/Cas 9 gene editing can convert risk alleles to protective alleles

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CONCLUSIONS1-GENETICS PLAY A MAJOR ROLE IN

ACQUIRING BMD AND FRACTURES

rare mutations provide huge insights

2-BONE REMODELING IS AN ACTIVE PROCESS WITH SIGNALS GOING AMONG ALL CELL TYPES

3-THE BIOENERGETICS OF BONE REMODELING ARE CRITICAL FOR FATE DETERMINATION AND BONE MASS

4- CLINICAL TO BASIC BACK TO CLINICAL IS A BONE PARADIGM