Spontaneous fractures in the mouse mutant sfx are caused by deletion of the gulonolactone oxidase gene, causing vitamin C deficiency.
Using a mouse mutant that fractures spontaneously and dies at a very young age, we identified that a deletion of the GULO gene, which is involved in the synthesis of vitamin C, is the cause of impaired osteoblast differentiation, reduced bone formation, and development of spontaneous fractures.


BACKGROUND
A major public health problem worldwide, osteoporosis is a disease characterized by inadequate bone mass necessary for mechanical support, resulting in bone fracture. To identify the genetic basis for osteoporotic fractures, we used a mouse model that develops spontaneous fractures (sfx) at a very early age.


METHODS
Skeletal phenotype of the sfx phenotype was evaluated by DXA using PIXImus instrumentation and by dynamic histomorphometry. The sfx gene was identified using various molecular genetic approaches, including fine mapping and sequencing of candidate genes, whole genome microarray, and PCR amplification of candidate genes using cDNA and genomic DNA as templates. Gene expression of selected candidate genes was performed using real-time PCR analysis. Osteoblast differentiation was measured by bone marrow stromal cell nodule assay.


RESULTS
Femur and tibial BMD were reduced by 27% and 36%, respectively, in sfx mice at 5 weeks of age. Histomorphometric analyses of bones from sfx mice revealed that bone formation rate is reduced by >90% and is caused by impairment of differentiated functions of osteoblasts. The sfx gene was fine mapped to a 2 MB region containing approximately 30 genes in chromosome 14. By using various molecular genetic approaches, we identified that deletion of the gulonolactone oxidase (GULO) gene, which is involved in the synthesis of ascorbic acid, is responsible for the sfx phenotype. We established that ascorbic acid deficiency caused by deletion of the GULO gene (38,146-bp region) contributes to fractures and premature death because the sfx phenotype can be corrected in vivo by treating sfx mice with ascorbic acid and because osteoblasts derived from sfx mice are only able to form mineralized nodules when treated with ascorbic acid. Treatment of bone marrow stromal cells derived from sfx/sfx mice in vitro with ascorbic acid increased expression levels of type I collagen, alkaline phosphatase, and osteocalcin several-fold.


CONCLUSIONS
The sfx is a mutation of the GULO gene, which leads to ascorbic acid deficiency, impaired osteoblast cell function, and fractures in affected mice. Based on these and other findings, we propose that ascorbic acid is essential for the maintenance of differentiated functions of osteoblasts and other cell types.
Mohan
Subburaman
S
Molecular Genetics Division, Musculoskeletal Disease Center, Jerry L. Pettis Memorial VA Medical Center, Loma Linda, California, USA. Subburaman.Mohan@med.va.gov
Kapoor
Anil
A
Singgih
Anny
A
Zhang
Zhang
Z
Taylor
Tim
T
Yu
Hongrun
H
Chadwick
Robert B
RB
Chung
Yoon-Sok
YS
Chung
Yoon-Suk
YS
Donahue
Leah Rae
LR
Rosen
Clifford
C
Crawford
Grace C
GC
Wergedal
Jon
J
Baylink
David J
DJ
D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism* / Q000494:pharmacology; D001206:Ascorbic Acid Deficiency / Q000235:genetics; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D001842:Bone and Bones / Q000378:metabolism*; D002454:Cell Differentiation; D002874:Chromosome Mapping; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000378:metabolism; D003720:Densitometry; D005269:Femur / Q000473:pathology; D017102:Fracture Healing; D050723:Fractures, Bone; D017353:Gene Deletion; D016678:Genome; D005838:Genotype; D050544:L-Gulonolactone Oxidase / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008957:Models, Genetic; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D010024:Osteoporosis / Q000378:metabolism; D010641:Phenotype; D012313:RNA / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000166:cytology / Q000473:pathology; D013977:Tibia / Q000473:pathology; D014965:X-Rays
D017931:DNA Primers; D018076:DNA, Complementary; D012313:RNA; D050544:L-Gulonolactone Oxidase; D001205:Ascorbic Acid