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Genes and Immunity (2000) 1, 260–264  2000 Macmillan Publishers Ltd All rights reserved 1466-4879/00 $15.00 www.nature.com/gene

Linkage of human tumor necrosis factor-alpha to human osteoporosis by sib-pair analysis N Ota1,2, SC Hunt3, T Nakajima1, T Suzuki4, T Hosoi4, H Orimo4, Y Shirai2 and M Emi1 1

Department of Molecular Biology, Institute of Gerontology, Nippon Medical School, 1–396 Kosugi-cho, Nakahara-ku, Kawasaki 211–8533, Japan; 3Cardiovascular Genetics, Department of Medicine, University of Utah School of Medicine, 410 Chipeta way, Salt Lake City, UT, 84108, USA; 2Department of Orthopedics, Nippon Medical School, 1–1-5 Sendagi, Bunkyou-ku, Tokyo, Japan; 4 Tokyo Metropolitan Institute of Gerontology and Hospital, Itabashi-ku, 173–0015, Tokyo, Japan

Osteoporosis as well as osteopenia are common human conditions considered to result from the interplay of multiple genetic and environmental factors. Twin and family studies have yielded strong correlation between measures of bone mass and a number of genetic factors. Certain genes (eg, cytokines such as interleukin-1, interleukin-6, or tumor necrosis factor-alpha) are capable of regulating metabolism, formation, and resorption of bone; all processes that determine bone mass. We tested 192 sib-pairs of adult Japanese women from 136 families for genetic linkage between osteoporosis and osteopenia phenotypes and allelic variants at the tumor necrosis factor-alpha (TNFA) locus, using a dinucleotide-repeat polymorphism located near the gene. The TNFA locus showed evidence for linkage to osteoporosis, with mean allele sharing of 0.478 (P = 0.30) in discordant pairs and 0.637 (P = 0.001) in concordant affected pairs. Linkage with osteopenia was also significant in concordant affected pairs (P = 0.017). Analyses limited to the post-menopausal women in our cohort showed similar or even stronger linkage for both phenotypes. The results provide evidence that genetic variations within the TNFA locus or adjacent genes affect regulation of mineral metabolism in bone and some of them confer risk for osteoporosis in adult women. Genes and Immunity (2000) 1, 260–264. Keywords: tumor necrosis factor-alpha gene; bone mineral density; osteoporosis; osteopenia; microsatellite polymorphism; sib-pair linkage

Introduction Osteoporosis is characterized by low bone mass and deterioration of the micro-architecture of bone tissue, with a consequent increase in fragility and susceptibility to fracture. The most important predictor of fracture is bone mineral density (BMD), a measurement that reflects many genetic and lifestyle factors. Its predictive value is well supported by results of twin1 and family studies.2 Knowing the genetic risk factors for an individual would assist physicians with diagnosis, prevention, and therapy. Multiple endocrine and metabolic factors play a role in the process of maturation of bone and in bone loss that accompanies aging.3 Bone loss originates from an imbalance between the resorption and formation phase of bone remodeling. Since the pathogenesis of osteoporosis among patients is likely to be heterogeneous, a rational approach to an understanding of the genetic background Correspondence: M Emi, Department of Molecular Biology, Institute of Gerontology, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki 211-8533, Japan. E-mail: memi얀nms.ac.jp This work was supported in part by a grant from the Ministry of Health and Welfare of Japan, by the Novartis Foundation for Gerontological Research and by the Promotion and Mutual Aid Corporation for Private School of Japan. The SAGE program package (Release 2.2) used in the study was supported by US Public Health Service Resource Grant No. 1-P41-RR03655 from the National Center for Research Resources. Received 3 September 1999; revised 28 November 1999; accepted 4 December 1999

in each case would require a more definitive method of genetic analysis. To date, numerous cytokines known to regulate the micro-environment of bone have been implicated in osteoporosis on the basis of association studies in human populations and phenotypes of mice in ‘knock-out’ experiments. Among the suspect genes are interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factoralpha (TNFA). In the study reported here, we chose to investigate the TNFA gene for possible effects of variations at this locus with respect to bone metabolism. We determined the relevant TNFA genotypes of 192 sib-pairs of Japanese women, and tested them for linkage to osteoporosis or osteopenia.

Results The 297 Japanese women of our panel, comprising 192 sib-pairs from 136 families, were genotyped for a CArepeat microsatellite present at TNFA locus. The size of the PCR products from this cohort of individuals ranged from 11 to 22 repeats of the dinucleotide. The distribution of these alleles in our test population, all of whom lived within a defined geographical region, is shown in Table 1. The results of qualitative sib-pair linkage analysis of osteoporosis/osteopenia against allelic variants at the TNFA locus are shown in Table 2a. Significant linkage of this locus to osteoporosis was indicated: mean allelesharing among the 53 discordant pairs (one affected, one

Sib-pair linkage of TNFA to osteoporosis N Ota et al

Table 1 Distribution of alleles of microsatellite at the TNFA locus among 297 Japanese women Alleles A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12

Size (bp)

Frequency

133 131 129 127 125 123 121 119 117 115 113 111

0.01 0.13 0.01 0.07 0.08 0.27 0.01 0.01 0.02 0.09 0.29 0.01

not) tended to be less than expected (0.478 ± 0.305) and was greater than expected among the 36 concordant affected pairs (0.637 ± 0.238, P = 0.001), on the basis of allele frequencies that had been estimated for the Japanese population as a whole. Mean allelic-sharing for osteopenia among the 63 discordant pairs was 0.501 ± 0.334 and among the 65 concordant affected pairs was 0.568 ± 0.251 (P = 0.017). Results of linkage analysis using only the post-menopausal women remained significant even though the number of pairs was reduced (P = 0.001, Table 2b). We also combined information from the three classes of sib-pairs by linear regression, to relate osteoporosis/osteopenia to the degree of allele sharing. When the concordance or discordance of disease status were regressed against the degree of allele sharing for all sibpairs, the regression lines for the polymorphism at the TNFA locus showed negative slopes which did not quite reach statistical significance (Table 3). The results became significant for osteoporosis when only post-menopausal women were analyzed (P = 0.006).

Discussion A relationship between the immune system and bone resorption was recognized nearly 25 years ago, with the first description in 1974 of ‘osteoclast activating factor’

(OAF) and other molecules produced by cells of the immune system.4 It has become clear since then that several molecular entities produced by cells of the immune system can affect differentiation and maturation of osteoblasts or osteoclasts in vitro. The most widely studied of these cytokines are interleukin-1 (IL-1), interleukin-6 (IL6) and TNFA. TNFA is involved in tumor-induced bone resorption5,6 and non-tumor-induced osteopenia7 and along with IL-1, is secreted from mononuclear cells of post-menopausal women.8 TNFA, a multi-functional cytokine, is essential for regulating the immune response, hematopoiesis, and bone resorption; in bone it stimulates formation of osteoclasts by including either proliferation of osteoclast-precursor cells or activation of differentiated osteoclasts,9 especially when triggered by estrogen deficiency.10 Various other genes have been suggested as candidates for the determination of BMD or for the pathogenesis of osteoporosis. For example, genetic variations have been examined at the vitamin D receptor locus;11 the estrogen receptor alpha locus12,13 or the collagen type I alpha 1 locus14 as to their roles in bone metabolism. We chose to test the TNFA locus by the method of sib-pair linkage analysis and genotyped a large panel of Japanese women for polymorphisms of a nearby microsatellite. We found that this locus was linked to osteoporosis, ie, to decreases in bone mineral density. In view of the increased estimated allele sharing in concordant pairs and a decreased sharing in discordant pairs, we conclude that the TNFA locus may be an important susceptibility gene for determination of BMD. The present study represents the first report of linkage of osteoporosis to the chromosomal region 6q21.3, containing the TNFA gene. When we analyzed separately the women of our panel who were post-menopausal, the results did not change with respect to statistically significance. The fact that menopausal status did not confound the linkage results was an important finding, although the number of pre-menopausal women was small. The synthesis of TNFA is known to be regulated at the transcriptional level.15 The TNFA promoter has been characterized and contains a number of important regulatory elements that effect TNFA transcription in response to various stimuli.16,17 Polymorphism of the 5′-

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Table 2 Sib-pair linkage of TNFA locus to osteoporosis and osteopenia Status

Pairs

0 1 2

84 (45) 53 (63) 36 (65)

0 1 2

54 (21) 47 (53) 36 (63)

Mean

s.d.

t-value

P-value

0.178 (−0.134) 0.535 (−0.027) 3.45 (2.17)

0.430 (0.553) 0.297 (0.511) 0.001* (0.017*)

(b) Post-menopausal women with osteoporosis (osteopenia) 0.548 (0.554) 0.305 (0.321) 1.15 (0.767) 0.451 (0.504) 0.302 (0.331) 1.12 (−0.792) 0.639 (0.563) 0.239 (0.254) 3.48 (1.95)

0.128 (0.226) 0.134 (0.531) 0.001* (0.027*)

(a) All women with osteoporosis (osteopenia) 0.506 (0.494) 0.293 (0.278) 0.478 (0.501) 0.305 (0.334) 0.637 (0.568) 0.238 (0.251)

Status: 0, concordant unaffected pairs; 1, discordant pairs; 2, concordant affected pairs. Criterion for osteoporosis diagnosis was a BMD ⬍70% of the mean among young adult females. Numbers in parenthesis represent an osteopenia diagnosis defined as a BMD ⬍80% of the mean among young adult females). A significant increase (status = 0.2) or decrease (status = 1) in allele sharing (compared to the expected 0.5) is taken as evidence for linkage. *Statistical significance of P values ⬍0.05. Total number of sib-pairs recruited in the present study was 192. Not all subjects had genotypes unambiguously determined for each marker, and therefore the total number of sib-pairs presented in the table varied among markers. Genes and Immunity


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Table 3 Regression of TNFA marker sharing identical-by-descent on concordance and discordant pairs Affected status

Effective D.F.

t-value

P-value

Intercept

Slope

the structure of the entire genomic region containing the TNFA gene and its control elements in affected individuals.

Materials and methods (a) All women osteoporosis osteopenia

154 154

(b) Post-menopausal women osteoporosis 125 osteopenia 125

1.41 0.795

0.080 0.214

0.396 0.417

−0.171 −0.101

2.56 1.09

0.006* 0.138

0.531 0.471

0.348 −0.155

A significantly negative regression line is taken as evidence for linkage. *Statistically significance of P values ⬍ 0.05.

franking region of the TNFA gene, responsible for transcriptional region of TNFA production, has been reported.18,19 Some evidence for the role of the region encompassing −308 in the transcription of TNFA has been provided by several researchers.20,21 Such variations in the gene expression might affect the production level of TNFA by osteoblast and /or osteoclast, leading to alteration in bone density. Other examples of genetic linkage have been known between several autoimmune diseases and TNFA polymorphism.22,23 Indeed, the polymorphisms within the TNFA gene promoter are associated with a particular clinical subtype of systemic lupus erythematosus, cerebral malaria, septic shock, rheumatoid arthritis or preterm premature rupture,24–28 although frequencies of these specific alleles are low in Japanese.29 The data presented here suggest that an allelic variation or mutation in, or adjacent, to the TNFA gene may affect bone metabolism and eventually cause a change in BMD. Future studies utilizing association tests may allow further genetic definition of this candidate region. To clarify the contribution of TNFA to the etiology of osteoporosis and osteopenia, it will be necessary to extend these studies to different population and to investigate

Subjects DNA samples were obtained from peripheral blood of 297 Japanese women from 136 families, comprising 192 sib-pairs. All lived in the Gunma area in the central part of Honshu mainland of Japan, and their ages ranged from 25 to 86 (mean, 64.0 ± 9.7). All were volunteers and gave their informed consent prior to the study. Measurement and criteria of bone mineral density (BMD) The BMD of radial bone at distal one-third (expressed in g/cm2) of each participant was measured by dual energy X-ray absorptiometry (DTX-200, Osteometer Ltd, Denmark). For our purposes, osteopenia was defined as a decrease of bone mineral density below 80% of the mean in young adult women (cut-off value, 0.381 = −1.8 s.d.), Osteoporosis was defined as a decrease of bone mineral density below 70% of the mean in young adult women (cut-off value, 0.333 = −2.6 s.d.), according to general criteria recommended by the Japanese Society for Bone and Mineral Research.30 The BMD of radial bone (expressed in g/cm2) of each participant was measured by dual energy X-ray absorptiometry (DPX-L, Lunar Co, USA). This parameter was recorded as adjusted BMD, in order to correct for differences in age, height, and weight. The formulas were as follows: Body mass index (BMI) = (body weight) (kg)/(body height)2 (m), adjusted BMD = BMD −0.0052 × (73.2 − age) + 0.0088 × (23.2 − BMI). Figure 1 shows the distribution of radial BMD with

Figure 1 Distribution of radial BMD with regard to age group for participants in the present study and general Japanese. BMD was measured by dual X-ray absorptiometry (DTX-200). Genes and Immunity


regard to age groups of the participants in the present study and the distribution in the general Japanese population published previously.30 Although many researchers measure BMD in the lumbar spine, we measured BMD in the radius, following the Guideline for Osteoporosis Screening in a health checkup program conducted by the Ministry of Health and Welfare of Japan.30 This method is recommended for measuring BMD in elderly females, in whom osteoporosis is often accompanied by ortho-arthritis of the spine. Apparently higher average of BMD might be due to such differences in methods of measurement. Determination of microsatellite polymorphism by PCR PCR amplification of the CA-repeat microsatellites at the TNFA locus was performed in a volume of 10 ␮l containing 20 ng of genomic DNA obtained from peripheral blood, 10 mm of Tris HCl (pH 8.4), 50 mm of KCl, 1.5 mm of MgCl2, 0.01% of gelatin, 200 ␮m of each dNTP, and 0.25 units of Taq polymerase, and each set of 2.5 pmol of [32P] end-labeled forward primer and non-labeled reverse primer that we have described previously. Cycle conditions were 94°C for 4 min, then 30 cycles of 94°C for 30 sec, 60 or 65°C for 30 sec, and 72°C for 30 sec, with a final extension step of 5 min at 72°C in a Gene Amp PCR9600 System (Perkin–Elmer Cetus, Norwalk, CT, USA). PCR products were electrophoresed for 2 h at 2000 V in 0.3-mm thick denaturing 6% polyacrylamide gels containing 36% formamide and 8 m urea. Gels were transferred to filter papers, dried at 80†C, and autoradiographed. The size of each allele was determined by comparison with a sequencing ladder of control DNAs.31 Statistical analysis In qualitative-trait analysis using osteoporosis as affected status, we analyzed three classes of sib-pairs; (1) both sibs unaffected (clinically concordant unaffected sib-pairs); (2) one sib affected and the other not (clinically discordant sib-pairs); (3) both sibs affected (clinically concordant affected sib-pairs). Non-parametric linkage analysis was performed using the SIBPAL program (version 2.7) of the SAGE package.32 A significant increase in allele-sharing (⬎0.5) for concordant pairs, and/or a significant decrease in allele sharing (⬍0.5) for discordant pairs was considered evidence for linkage. Although the total number of sib-pairs recruited in the present study was 192, not all subjects had genotypes unambiguously determined for the marker. Because menopausal status could have profound effects on sib-pair phenotypic concordance, we repeated all analyses using only those women who were over age 55. This resulted in 113 families, with a total of 141 sib-pairs. This program applies the algorithm with the given allele frequencies appropriate for random samples, and estimates the proportion of alleles the sib pair shares identity by state at that locus.

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