Quantification of vitamin D receptor mRNA by competitive polymerase chain reaction in PBMC: lack of correspondence with common allelic variants.
It has been recently claimed that polymorphism for the vitamin D receptor (VDR) influences several aspects of calcium and bone metabolism. To evaluate the physiologic plausibility of these claims, we compared the abundance of the VDR mRNA in peripheral blood mononuclear cells (PBMCs) between different VDR genotypes using a quantitative reverse transcribed polymerase chain reaction-based method. The method is based on the coamplification of VDR cDNA and an internal standard consisting of known concentrations of a human VDR CDNA mutated at a BglII restriction site; the interassay coefficient of variation is 11%. To validate the method, we made use of earlier receptor binding studies indicating that normal human monocytes and activated, but not resting, lymphocytes expressed the VDR. The concentration of the VDR mRNA was 10(-8) to 10(-7) g/g of total RNA in cell-sorted monocytes and in in vitro activated lymphocytes, but only 10(-12) g/g of total mRNA in resting lymphocytes, establishing that the VDR mRNA determined by our method in PBMCs is due to constitutive expression in monocytes. Following an initial genotype screening of 85 normal volunteers by polymerase chain reaction or restriction fragment length polymorphism analysis, 14 individuals with the Bb genotype, 12 with the bb genotype, and 12 with the BB genotype were selected. The concentration of the VDR mRNA, corrected for the number of monocytes, was similar among the three genotype groups, as were the other variables examined: serum calcitriol, serum osteocalcin, and vertebral and hip bone density. We conclude that VDR polymorphism does not affect the abundance of the VDR mRNA.
Mocharla
H
H
Department of Medicine, The University of Arkansas for Medical Sciences, Center for Osteoporosis and Metabolic Bone Diseases, Little Rock 72205, U.S.A.
Butch
A W
AW
Pappas
A A
AA
Flick
J T
JT
Weinstein
R S
RS
De Togni
P
P
Jilka
R L
RL
Roberson
P K
PK
Parfitt
A M
AM
Manolagas
S C
SC
D000368:Aged; D000369:Aged, 80 and over; D000483:Alleles; D005260:Female; D005434:Flow Cytometry; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry*; D008213:Lymphocyte Activation; D008297:Male; D008875:Middle Aged; D010835:Phytohemagglutinins / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000097:blood*; D018167:Receptors, Calcitriol / Q000235:genetics*; D015203:Reproducibility of Results; D014158:Transcription, Genetic
D010835:Phytohemagglutinins; D012333:RNA, Messenger; D018167:Receptors, Calcitriol