Yasumasa Shirai

A nucleotide variant in the promoter region of the interleukin-6 gene associated with decreased bone mineral density

AbstractInterleukin-6 (IL6) has come to be regarded as a potential osteoporotic factor because it has stimulatory effects on cells of the osteoclast lineage, and, thus, may play a role in the pathogenesis of bone loss associated with estrogen deficiency. We previously described association of the IL6 microsatellite with bone mineral density (BMD), as well as genetic linkage of the IL6 locus to human osteoporosis, by means of sib-pair analysis. However, the molecular mechanism by which this locus regulates BMD remains unknown. Accordingly, we searched for polymorphisms in the 5′ and 3′ flanking regions and in all five exons of the IL6 gene in a Japanese population sample. We identified three single-nucleotide sequence variations: a C/G substitution at nucleotide (nt) −634 in the promoter region, a G/A substitution at nt 4391 in the 3′ noncoding region, and a variation in the AnTn tract around nt −447. The last of these had already been observed in Caucasians, as well as in Japanese. The single-nucleotide polymorphism at −634 created a restriction site for the BsrBI endonuclease, and the frequency of the minor (G) allele was 0.184. Five haplotypes were constructed among three variations examined in the population. Linkage disequilibrium was observed between the variation at −634 and the variation at 4391, as well as between the variation at −634 and the AnTn tract variation. We found a significant correlation, in 470 subjects, between the presence of the G allele and decreased BMD, by analysis of variance. When BMD values were compared among the three genotypic groups (G/G, G/C, C/C) at nt −634, BMD was lowest among the G/G homozygotes (mean ± SD; 0.284 ± 0.062 g/cm2), highest among the C/C homozygotes (0.314 ± 0.059 g/cm2), and intermediate among the heterozygotes (0.303 ± 0.066 g/cm2; P < 0.05).Given the several lines of evidence from different genetic studies, we suggest that IL6 is, indeed, one of the genes affecting bone metabolism, in which variations can lead to osteoporosis.

Histochemical Changes in the Multifidus Muscle in Patients With Lumbar Intervertebral Disc Herniation

Study Design. The histochemical changes in the multifidus muscle in 29 patients with L4–L5 lumbar intervertebral disc herniation were studied. Objectives. To clarify how nerve root impairment affects the histochemical properties of the lumbar multifidus muscle in patients with lumbar intervertebral disk herniation. Summary of Background Data. There have been several studies on histochemical changes in lumbar muscles in patients with nerve root impairment, but the findings concerning changes in muscle fiber sizes vary among investigators. Methods. Biopsy specimens were obtained intraoperatively from the L4 and L5 bands of the multifidus muscle on the affected and nonaffected sides. The specimens were stained with ATPase to evaluate the size of the fibers and structural changes. Results. In the L5 muscle band, the mean sizes of Type 1 and Type 2 fibers on the affected side were significantly smaller than those on the nonaffected side (Type 1:P < 0.01, Type 2:P < 0.001). The decrease in size was 6.4% for Type 1 and 9.8% for Type 2. Increased percentages of Type 1 fibers and a high incidence of small angular fibers and fiber type grouping were also shown on the affected side. In contrast, in the L4 muscle band, no side-to-side differences in the histologic findings were observed. There was no significant level-to-level difference in the mean size of Type 1 or Type 2 fibers on either the affected or the nonaffected side. Conclusions. These results suggest that nerve root impairment leads to atrophy of Type 1 and Type 2 fibers, with structural changes in the multifidus muscle only at the involved level.

Sensory nerve supply in the human subacromial bursa.

The subacromial bursa is the major component of the subacromial gliding mechanism. The neural elements of the subacromial bursa obtained from specimens that underwent autopsy and surgery were investigated by the silver impregnation and immunohistochemical methods with antisera to substance P and calcitonin gene-related peptide; which are considered to be involved in nociceptive transmission, and protein gene product 9.5. Free nerve endings, Ruffini endings, Pacinian corpuscles, and two kinds of unclassified nerve endings were observed. Most of these receptors were observed of the roof side of the coracoacromial arch, which is exposed to stress because of the impingement. A delta and C fibers, thought to be nerve fibers of free nerve endings, were immunoreactive to substance P and calcitonin gene-related peptide. On the other hand, thick fibers thought to originate in encapsulated mechanoreceptors were not immunoreactive to substance P. The subacromial bursa receives nociceptive stimuli and proprioception and seems to regulate appropriate shoulder movement.

Expression of bone morphogenetic proteins and rat distal-less homolog genes following rat femoral fracture.

Abstract: Expression of the genes encoding bone morphogenetic proteins (BMPs), BMP type IA receptor (BMPR-1A), and rat distal-less homolog (rDlx) was studied in bone, callus, and the surrounding soft tissue following rat femoral closed fracture, using RT-PCR-based techniques. Before fracture, the genes encoding BMP-5, BMP-6, and BMPR-1A were found to be expressed in both bone and the surrounding soft tissue, whereas the BMP-2 gene was expressed only in bone and BMP-7 was not expressed in either tissue. Expression of these genes was unaffected by fracture. The gene encoding BMP-4 was also expressed in both bone and the surrounding soft tissue before fracture. Moreover, although unchanged in bone, 6 h after fracture BMP-4 expression was increased tenfold in the surrounding soft tissue. The increased BMP-4 expression was transient and returned to prefracture levels within 72 h. Expression of rDlx was also increased in bone after fracture, but at later times than were observed with BMP-4: elevated rDlx expression was detected after 48 h and persisted for 30 days or more. No expression of rDlx was observed in the surrounding soft tissue before or after fracture. These findings indicate that BMP-4 and rDlx are selectively expressed following femoral fracture in the rat, and also suggest that they are involved in the formation of the callus at an early point during the postfracture healing of bone.

Ethnic Difference in Contribution of Sp1 Site Variation of COLIA1 Gene in Genetic Predisposition to Osteoporosis

Abstract. Osteoporosis, a condition characterized by low bone mineral density (BMD) leading to bone fragility [1], is a major public health concern in Japan as well as in other countries. Although genetic predisposition seems to be a factor in the pathogenesis of osteoporosis [2–4], the precise cohort of genes that may be involved is not well defined. The COLIA1 and COLIA2 genes encode polypeptide constituents of collagen type Iα1 and Iα2, respectively. Both are important candidates as genetic regulators of BMD, since mutations in either gene result in osteogenesis imperfecta, a disorder characterized by severe osteoporosis [5]. Some patients with adult osteoporosis also carry mutations in COLIA1 or COLIA2 genes [6].

Linkage of human tumor necrosis factor-alpha to human osteoporosis by sib-pair analysis

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.

A highly polymorphic CA repeat marker at the human tumor necrosis factor alpha (TNFAα) locus

AbstractTumor necrosis factor alpha (TNFα) in activated monocytes exerts cytotoxic activity and has a variety of other biological effects. We isolated a polymorphic dinucleotide (CA) repeat sequence from a genomic clone containing the gene located at 6p21.3. High heterozygosity (0.80) makes this polymorphism a useful marker in the genetic study of disorders affecting immunological response and cell differentiation.

A highly polymorphic CA repeat marker at the human interleukin 6 receptor (IL6R) locus

AbstractA polymorphic dinucleotide (CA) sequence was isolated from a genomic clone containing the human interleukin 6 receptor (IL6R) gene. High heterozygosity (0.81) makes this polymorphism a useful marker in the genetic study of disorders affecting the inflammation process and bone resorption.

ISOLATION AND RADIATION HYBRID MAPPING OF A HIGHLY POLYMORPHIC CA REPEAT SEQUENCE AT THE HUMAN NUCLEAR FACTOR KAPPA-BETA SUBUNIT 1 (NFKB1) LOCUS

AbstractThe transcriptional factor nuclear factor kappa-beta (NFKB) consists of a multicomponent protein complex that plays a major role in the regulation of many viral and cellular genes. The NFKB complex has two alternative DNA binding subunits. We isolated a polymorphic dinucleotide (CA) repeat sequence from a genomic clone containing the NFKB subunit 1 (NFKB1) gene located at 4q23-24. High heterozygosity (0.813) makes this polymorphism a useful marker in the genetic study of disorders affecting the immune response and cell differentiation.

A highly polymorphic CA repeat marker at the interleukin-11 locus.

The interleukin-11 (IL-11) stimulates T cell-dependent development of immunoglobulin-producing B cells and collaborates with IL-3 in supporting murine megakaryocyte colony formation. The interleukin-11 (IL-11) also stimulates osteoclast formation and inhibits osteoclast function in vitro and has been implicated in estrogen deficiency-induced bone loss. We isolated a polymorphic dinucleotide (CA) repeat sequence from a genomic clone containing the IL-11 gene located at 19q13.3–q13.4. High heterozygosity (0.81) makes this polymorphism a useful marker in genetic study of disorders affecting immune response and bone metabolism.