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Molecular Biology Reports

, Volume 46, Issue 2, pp 1667–1674 | Cite as

Epigenetic regulation of BMP2 gene in osteoporosis: a DNA methylation study

  • Mehrunnisa M. Raje
  • Richa AshmaEmail author
Original Article

Abstract

Osteoporosis is a multifactorial disease in which genetic factors and epigenetic modifications play a major role. DNA methylation is known for gene silencing and its effect on BMP2 promoter has been studied here to understand its regulatory activity in osteoporosis pathogenicity. CpG methylation in the BMP2 promoter was analyzed by performing bisulfite specific PCR on the gDNA samples extracted from whole blood of osteoporotic (n = 24) and healthy (n = 24) individuals. Disproportionate allele frequency of CpG sites was calculated statistically. Differential BMP2 expression was estimated using quantitative RT-PCR technique. Luciferase reporter assay was performed to determine and confirm differential transcriptional activity of BMP2 promoter due to methylation. Total of 14 CpG sites were reporter in the BMP2 promoter of which, CpG site at − 267th position upstream to TSS was found to have disproportionate allele frequency among osteoporotic and healthy individuals and was found to be significantly associated with osteoporosis condition. Functional and gene expression analysis of this methylated site using luciferase reporter vector and Real Time PCR approach, suggested reduced transcriptional activity of BMP2 promoter as well as decreased gene expression in disease condition. BMP2 is being a central signaling molecule, aberrant methylation in the promoter region may result into down regulation of osteoblast markers involved in bone formation.

Keywords

Osteoporosis Epigenetics BMP2 DNA methylation Luciferase reporter assay 

Abbreviations

ALP

Alkaline phosphatase

BMD

Bone mineral density

BMP2

Bone morphogenetic protein 2

BMP2K

BMP2 inducible kinase

BMP4

Bone morphogenetic protein 4

BMP6

Bone morphogenetic protein 6

BMP7

Bone morphogenetic protein 7

cDNA

Complementary DNA

CI

Class interval

CpG

5′—C—Phosphate—G—3

Dlx5

Distal-less homeobox 5

DNA

Deoxyribonucleic acid

DXA

Dual X-Ray absorptiometry

ECACC

European Collection Of Authenticated Cell Cultures

EDTA

Ethylenediaminetetraacetic acid

E-MEM

Eagle’s Minimum Essential Medium

EMP2

Epithelial membrane protein 2

ERKs

Extracellular signal-regulated kinases

FC

Fold change

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

gDNA

Genomic DNA

ICMR

Indian Council of Medical Research

ID

Identity

LA

Luria agar

LB

Luria Broth

LD

Linkage disequilibrium

mRNA

Messenger RNA

MSA

Multiple sequence alignment

NCBI

National Center for Biotechnology Information

OSX

Osterix

PBMC

Peripheral blood mononuclear cell

PBS

Phosphate-buffered saline

PCR

Polymerase chain reaction

qRT-PCR

Quantitative real time polymerase chain reaction

RefSeq

Reference sequence database

RLU

Relative luciferase activity

RNA

Ribonucleic acid

RR

Risk ratio

RT-PCR

Reverse transcriptase polymerase chain reaction

RUNX2

Runt related transcription factor 2

SD

Standard deviation

SNP

Single nucleotide polymorphisms

TGF-Beta

Activated kinase 1

TF

Transcription factor

TSS

Transcription start site

WHO

World Health Organization

Notes

Compliance with ethical standards

Conflict of interest

The authors have declared that no conflict of interest exists.

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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Center for Advanced Studies, Department of ZoologySavitribai Phule Pune UniversityPuneIndia

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