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Association of Collagen Type 1 1 Gene Polymorphism with Bone Density in Early Childhood¹

Departments of Radiology (J.S., V.G.) and Pediatrics (J.M.V.T., F.K.), Childrens Hospital Los Angeles; Los Angeles, California 90027; Department of Biostatistics (J.S.), University of California Los Angeles School of Medicine, Los Angeles, California, 90024

Address correspondence and requests for reprints to: Vicente Gilsanz, M.D., Radiology Department, Childrens Hospital Los Angeles, 4650 Sunset Boulevard, M.S.#81, Los Angeles, California 90027. E-mail: gilsanz{at}hsc.usc.edu

	Abstract

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Osteoporosis is a disease characterized by the development of nontraumatic fractures, most commonly in the vertebrae of elderly women. Approximately 500,000 elderly women in the United States are newly diagnosed with vertebral fractures every year, as the compressive strength of the vertebra, mainly determined by the density of cancellous bone and its cross-sectional area, declines with age. A recent study in women suggested that a polymorphism in the Sp1 binding site of the collagen type I gene (COLIA1) was related to decreased vertebral bone mass and vertebral fractures. Determining the phenotypic trait(s) responsible for this relationship and whether this association is manifested in childhood would further define the structural basis for decreased bone mass and help identify children "at risk" for fractures later in life. We therefore studied the COLIA1 gene polymorphism and measurements of the size and the density of vertebral bone in 109 healthy, prepubertal girls. On average, 22 girls with the Ss genotype and one girl with the ss genotype had 6.7% and 49.4% lower cancellous bone density in the vertebrae than girls with the SS genotype. In contrast, there was no association between the size of the vertebrae and the COLIA1 genotypes.

	Introduction

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OSTEOPOROSIS is a complex disease characterized by low bone mass and the development of nontraumatic fractures, most commonly in the vertebrae (1). Because it is now clear that this disease, which is largely irreversible once it has manifested, has its antecedents in childhood, defining the genetic and environmental factors responsible for variations in bone mass during skeletal growth should aid in the design of early interventions to prevent osteoporosis (2).

Although at least 70% of bone mass is genetically determined, unraveling the genes responsible for the phenotypic variability of this skeletal trait has proven to be a notoriously elusive task. For the past several years, we have examined the influence that candidate genes, reported to be associated with adult bone mass, have on the phenotypic variability of skeletal development in children. The first such gene to be so identified was the vitamin-D receptor gene, which accounts for a relatively small proportion of the genetic effect on bone mass, both in children and adults (3, 4, 5). More recently, it was suggested that a polymorphism in the Sp1 binding site of the collagen type I 1 gene (COLIA1) was related to decreased vertebral bone mass and vertebral fractures in women (6, 7). To determine whether this association is manifested in childhood, we studied the COLIA1 genotypes and quantitative computed tomography (CT) measurements of the two components of bone mass, the volume and the density of bone, in healthy girls.

	Subjects and Methods

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Study subjects

The study subjects were 109 healthy, prepubertal, Mexican-American girls, ages 6.7–12.3 yr, who were recruited from schools of Los Angeles County. A large subset of this population was previously analyzed to study the relationship between vitamin-D receptor gene polymorphism and bone density. The protocol for the current study was approved by the Childrens Hospital of Los Angeles Institutional Review Board, and informed consent was obtained from all subjects and parents.

Candidates for the study were excluded if they had a diagnosis of chronic illness, had been ill for longer than 2 weeks during the previous 6 months, had been hospitalized at any time since birth, or had taken any medications, vitamin preparations, or calcium supplements regularly within the previous 6 months. Candidates were also excluded if their parents or grandparents were not of Mexican origin.

All children underwent a physical examination by a pediatric endocrinologist to determine their stage of sexual development. Only candidates who were prepubertal and whose height and weight were within the 5th and 95th percentiles for the mean age-adjusted normal values were enrolled in this study. Body-surface area and body-mass index were calculated as previously described (8). Skeletal maturation was assessed according to the method of Greulich and Pyle (9), and girls in whom chronological and bone age differed by more than 1 yr were also excluded.

Bone measurements

All CT bone measurements were obtained with the same scanner (CT-T 9800; General Electric Co., Milwaukee, WI) and mineral reference phantom (CT-T bone densitometry package; General Electric). Cancellous bone density and cross-sectional area were measured at the midportion of the L1 through L3 vertebral bodies, as previously described (10, 11). Coefficients of variation for determinations of vertebral cancellous bone density and cross-sectional areas have been determined as 2% and 0.6%, respectively (5).

Genotype analysis of a G-T polymorphism of the COLIA1 gene

The genotype for the polymorphism of the COLIA1 gene was determined by PCR amplification of a 255 bp fragment of the regulatory region and enzymatic digestion of the product with BalI. Primers, which introduce a restriction site for BalI in the polymorphic allele, have been described previously (6). PCR was performed with a Biometra Trio thermoblock (Biometra Biomedizinische Analytik GmbH, Gottingen, Germany) using standard conditions, 40 cycles and 67 C as annealing temperature. The genotype was defined as S or s according to the absence or presence of restriction site, respectively. Products were digested overnight with BalI (Promega Corp.) and resolved by agarose gel electrophoresis.

Biochemical assessment

After an overnight fast, blood was taken for routine serum chemistry, calciotropic hormones, and markers of bone turnover. Intact parathyroid hormone, 25-hydroxyvitamin D (25(OH)D), 1,25-dihydroxyvitamin D (calcitriol), alkaline phosphatase, bone specific alkaline phosphatase, and osteocalcin were analyzed by Corning Nichols Institute Diagnostics (San Juan Capistrano, CA).

Statistical analysis

All results are expressed as mean ± 1 SD. The data were analyzed by using Student’s t-test for unpaired samples (12). All tests were two-sided, and P values of less than 0.05 indicated statistical significance for a power of 80%.

	Results

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The anthropometric characteristics of the subjects studied are shown in Table 1. By design, the height and weight of each girl was within the 5th and 95th percentiles for the mean age-adjusted normal values, and chronological age and bone age were within 1 yr. No differences were found in the developmental parameters of the girls within the COLIA1 genotypes. The most common COLIA1 genotype was SS (81.6%), while the ss genotype was present in only one girl.

View larger version (11K): [in this window] [in a new window]   Figure 1. Cancellous bone density of the vertebrae in relation to COLIA1 Sp1 polymorphism genotypes in 109 prepubertal girls. Values are the median (line) interquartile range (box) and range of values (whiskers). SS, homozygous for absence of BalI site; ss, homozygous for presence of BalI site; Ss, heterozygous. P values are for differences between SS and Ss and ss genotypes using Student’s t tests.

	Discussion

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With respect to the population studied, two characteristics should be noted. First, the girls examined were of Mexican-American heritage, and previous studies suggest a large degree of genetic homogeneity in this population (13). The COLIA1 genotypes for the Sp1 alleles were in Hardy-Weinberg equilibrium, and their frequencies were similar to the ones found in British and Dutch populations (6). Second, we chose to examine prepubertal children to avoid the confounding effect that the pubertal growth spurt has on skeletal development (11, 14). Differences among COLIA1 gene alleles may have been more difficult to demonstrate if assessments had been made during puberty, when large increases in skeletal size, bone mass, and bone density occur over a brief period of time (11, 14).

The mechanisms by which the different COLIA1 alleles affect bone density have yet to be determined. Type 1 collagen is the major structural protein in bone and consists of a heterotrimeric complex of two 1 and one 2 molecules. The majority of patients with osteogenesis imperfecta have mutations in the gene for either the pro 1 (I) chain or the pro 2 (I) chain of type I procollagen. Our results raise the possibility that carriage of the s allele may be associated with a disturbance in the relative abundance of COLIA1 and COLIA2 messenger RNAs, as occurs in osteogenesis imperfecta (15). No statistically significant differences were found between the COLIA1 polymorphism and biochemical measurements for serum values of calciotropic hormones and bone turnover markers. Specifically, there were no differences between parathyroid hormone, 25-hydroxyvitamin D (25(OH)D), 1,25-dihydroxyvitamin D (calcitriol), alkaline phosphatase, bone specific alkaline phosphatase, and osteocalcin among genotypes (data not shown).

In conclusion, the results of this study indicate that the COLIA1 gene alleles are associated with the normal variations in the apparent density of cancellous bone in the axial skeleton of prepubertal girls. This information may contribute to the identification of a subset of the population of normal girls that may be at risk of developing vertebral fractures later in life and may ultimately be of value in the planning of early preventive strategies for osteoporosis.

	Acknowledgments

 
The authors would like to thank Ms. Cara L. Beck for her technical assistance and comments on this manuscript.

	Footnotes

 
¹ Supported in part by Grant R01-AR4–1853-01A1, from the National Institute of Arthritis and Musculoskeletal and Skin Diseases, and Grant 1RO1 LM06270–01, from the National Library of Medicine.

Received September 8, 1998.

Revised December 11, 1998.

Accepted December 28, 1998.

	References

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