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CHAPTER 8/GENETIC TRANSMISSION OF COMMON SPINAL DISORDERS / 103
of occupational physical loading from materials handling
and work postures. The study findings indicated that disc
degeneration may be explained primarily by familial
influences, which are most likely genetic, and as yet
unidentified factors, which may include complex interactions. This study provides a f irst estimate of the relative
importance of specif ic environmental agents and overall
familial influences, including genetic factors (34). The
remaining variance that is unaccounted for by the specific environmental and familial sources of variation is
due to measurement error and yet unknown environmental effects.
Classic Twin Studies of Disc Degeneration
Following the earlier studies suggesting the possibility
of a substantial genetic influence, Sambrook et al. conducted a classic twin study to examine the hypothesis that
disc degeneration has a major genetic component (35).
Spine MR images were obtained for 86 pairs of monozygotic twins and 154 dizygotic twins, 80% of who were
female, from Australian and British twin registries. A
substantial genetic influence on disc degeneration was
found. For an overall score of disc degeneration, comprised of disc height, signal intensity, bulging, and anterior osteophyte formation, heritability estimates were
74% [95% confidence interval (CI), 64% to 81%] for the
lumbar spine and 73% (95% CI, 64% to 80%) for the cervical spine. Heritability estimates were adjusted for age,
weight, smoking, occupation, and physical activity. An
analysis of individual MRI findings suggested that disc
bulging and height were the primary contributors to the
genetic determination of the disc degeneration summary
score. Interestingly, a genetic influence was not apparent
for signal intensity (35).
The findings of Sambrook et al. indicate a substantial
genetic influence. What is not known is whether specif ic
gene effects of relatively large magnitude exist or if the
genetic contribution is due to small effects of many genes
(35).
Direct Evidence of a Genetic Influence on Disc
Degeneration
Genetic influences on intervertebral disc degeneration
in humans were confirmed in 1998. In a study, using
spine MRI it was shown that low-signal intensity of thoracic and lumbar discs was associated with TaqI tt-genotypes of the vitamin D receptor gene. A similar pattern
was found between the summary scores of signal intensity, bulging, and disc height for both TaqI and FokI
genotypes (36). TaqI and FokI each accounted for a substantial portion (6% to 7%), of the inter-individual variance in disc degeneration as measured through signal
intensity. Another study using spine X-ra y found an association between Taq polymorphisms and the severity of
osteophytosis and presence of disc narrowing, and more
weakly, with the presence of osteophytosis (37).
A later investigation of the associations of vitamin D
receptor TaqI polymorphisms and spine degeneration
demonstrated that those with the tt genotype also had
more anular tears but less bulges and osteophytes than
those with the TT genotype (38). These f indings emphasized the need for caution in combining specific suspected degenerative phenomena into summary scores.
Also, the finding of the association between anular tears
and genetics could be of importance because anular tears
may be related to the pathophysiology of back pain. In
another study, multilevel and severe lumbar disc de generation was observed among 64 women with shorter variable numbers of tandem repeat length of the aggregate
gene (39). In addition, the 5A5A and 5A6A genotypes of
metalloproteinase-3 gene were associated with more
degenerative findings in elderly individuals than those
with the 6A6A genotype (40).
In animal studies, accelerated joint and intervertebral
disc degeneration were observed in transgenic mice
(COL9A1) based on X-rays and histologic methods. The
spinal changes included shrinkage of the nucleus pulposus, anular fissures and herniations, and slight osteophyte
formation (41). Howe v er, none of the known mutations in
COL9A1 have been associated with disc degeneration in
humans.
Although several gene forms associated with various
aspects of disc degeneration hav e already been identified,
it is likely that new gene forms associated with lumbar
degeneration, pathology, and symptoms will be found
over the coming years with the rapid growth in genetic
research.
Several mechanisms have been suggested through
which hereditary factors could influence disc degeneration and herniation. Genetic effects on the size and shape
of spinal structures could affect the spine’s mechanical
properties, and thus its vulnerability to external forces
(42). Biologic processes associated with the synthesis
and breakdown of the disc’s structural and biochemical
constituents could be genetically predetermined, in part,
leading to accelerated degenerative changes in some persons relative to others. The latter hypothesis has received
some support from the recent f indings of Annunen et al.
and Paassilta et al. who found mutations in two collagen
IX genes, COL9A2 and COL9A3, to be associated with
disc pathology and symptoms (25,27).
BACK PAIN
Much more than just structural variations need to be
considered in genetic and other determinants of back
pain. For example, genetic influences could affect pain
through a variety of mechanisms dealing with structural,
neurologic, inflammatory, other physiologic and behavioral characteristics.
104 /SECTION I/BASIC SCIENCE
The relative importance of genes versus experience in
human pain perception remains unclear; in animal studies there are significant individual differences in both
nociceptive and analgesic sensitivity. Yet such differences are not necessarily attributable to genetics. Most
often the familial aggregation of pain has been attributed to shared environmental influences and familial
modeling (43–46). MacGregor et al., in a classic twin
study of sensitivity to forehead pressure pain threshold,
found heritability estimates of only 10%, indicating that
shared family environmental factors may be signif icant
in pain thresholds (47).
Classic Twin Studies Suggesting Genetic
Susceptibility
Little is known about the role of genes in common lo w
back pain problems or the pathways or mechanisms
through which they ma y influence these problems. There
have, however, been a of couple classic twin studies suggesting a genetic component. Bengtsson and Thorson
investigated possible genetic influences on back pain in
a cohort of 5,029 monozygotic and 7,876 dizygotic
Swedish twin pairs (48). Back pain was defined as an
affirmative answer to the question, “Have you had so
much back pain during the last few years that you found
it difficult to work?” Such pain was reported by about
15% of this cohort of twins ranging from 15 to 47 years
of age. Pain concordance among twins with similar
physical work environments was higher among monozygotic (25%) than dizygotc (15%) twin pairs (except in
men performing light work), leading the authors to conclude that there is a relationship between genetic factors
and back pain (48). The Swedish data are reported by
gender, workload, and zygosity, but not by age, which is
unfortunate given the findings of Heikkilä et al. of a differential effect of heredity by age on sciatica and associated hospitalizations (24).
Another classic twin study using over 700 twin pairs
was presented by MacGregor et al. at an American College of Rheumatology meeting in 1999 (49). They found
a substantial genetic contribution to the occurrence of
severe back pain, with genetic factors accounting for
73% of the variance in population liability. They also
reported on a subset of 97 monozygotic and 234 dizygotic pairs that had MR images available for analyses
and found that more than 50% of the total genetic variance in back pain remained unexplained by genes
involved in MRI changes. This suggests that there are
other mechanisms through which genes may influence
back pain than simply through structural changes in the
disc. Mogil has noted that pain is considered both a sensation and an emotion, with considerable complexity
and subjectivity. Yet, pain is also being studied at the
level of the gene (50). The aforementioned studies
should motivate more studies of the roles and relative
contributions of cultural and genetic inheritance of back
pain.
OTHER SPINAL DISORDERS WITH GENETIC
CONTRIBUTIONS
Genetic contributions have been suggested or identified for a number of other spinal disorders, such as scoliosis, Scheuermann disease, spondylolysis, spina bifida,
and spinal stenosis. Several family studies indicate that
heredity has a role in scoliosis. Nearly identical “mirror
images” of congenital lumbar scoliosis for a brother and
sister, and two sets of identical twins with concordant
scoliotic curves have been reported (51). In one family
study from 1975, scoliosis appeared in 15 members of a
family in 3 generations (52).
Segregation analysis was applied to 101 pedigrees
from Russia with idiopathic scoliosis (more than 10°) and
to 90 pedigrees with Scheuermann disease. Using transmission probability models, a significant contribution of
one major causal gene was established and inheritance
could be described according to a dominant major gene
diallele model for both diseases. The authors concluded
that only the carriers of the mutant allele develop pronounced forms of the disease. For scoliosis, only 30% of
males and 50% of females with the mutant gene should
manifest the disease (53). All male carriers of the mutant
allele develop Scheuermann disease, while only half of
female carriers manifest the disease. The frequency of
scoliosis in the families with Scheuermann disease was
8%. The authors concluded that the “familial aggregation
of these two spinal patholo gies in the present sample may
indicate a genetic unity of Scheuermann disease and idiopathic scoliosis” (53,54).
Scoliosis can also be a consequence of other severe
diseases such as Marfan syndrome, familial dysautonomia, spondylocostal dysostosis, congenital lordoscoliosis
due to lumbar segmentation defects and incomplete formation of lumbar vertebrae, diastrophic dwarfism, and
familial Rett syndrome (55–57). The occurrence of scoliosis in the presence of other hereditary connective tissue
syndromes raises the possibility that idiopathic scoliosis
and congenital scoliosis are in fact a heterogeneous group
of disorders with varied pathogenetic mechanisms (58).
From a systematic revie w using different genome databases, there were three candidate loci for human scoliosis
(58). Genome-wide linkage surveys in large multiplex
families indicate concordantly a limited number of
genetic loci predisposing to idiopathic scoliosis: three
loci on chromosomes 6p, distal 10q, and 18q in one family and distal chromosome 10q on another (59). The role
of genetic factors in the development of scoliosis has
been well documented; however, reports of the specif ic
mode of genetic inheritance are inconclusive. These facts,
combined with the phenotypic variability of this disorder ,
suggest that the genetic expression of idiopathic scoliosis
CHAPTER 8/GENETIC TRANSMISSION OF COMMON SPINAL DISORDERS / 105
may be dependent upon multiple factors and genetic
interactions (60).
Spondylolysis also is suspected of having a genetic
component. Spondylolysis was found in 13% of young
Eskimos and in 74% of older Eskimos, a rate that is
higher in the older age group than in other ethnic groups,
leading Simper et al. to suspect a genetic influence (61).
Several family studies support that there is an inheritance
component in spondylolysis: 21% of descendants of a
male ancestor with spondylolysis also had the condition,
4% had also spina bifida occulta. This pedigree was consistent with autosomal-dominant inheritance and incomplete (about 75%) penetrance for spondylolysis (56). One
other survey identified 19% of relatives with spondylolysis (57). In addition, the reported cases of multiple lumbar spondylolysis could indicate the hereditary component (62–64). Spondylolysis can also be part of other
syndromes, such as osteopetrosis, where other findings
usually are clinically more important (65).
There are several reports about small case series indicating familial aggregation of spinal stenosis, commonly
associated with a narrow cervical canal and disc herniations and sometimes with other congenital anomalies
(66–69). Familial spinal canal stenosis has also been
associated with autosomal-dominant osteosclerosis, and
acrodysostosis (70). In addition, there are also case
reports about hypophosphatemic vitamin D–resistant
rickets as a cause of spinal canal stenosis (71).
SUMMARY
The study of genetic influences on common spinal disorders is rapidly progressing. Studies of familial aggregation were an initial step along this line of inquiry.
Familial aggregation, well beyond what would be expected by chance occurrence, has been found for outcomes such as hospitalizations for disc herniation in
juveniles and adults, sciatica, back pain, and disc degeneration. Familial aggregation also has been found to be
greater in younger than older subjects in the case of hospitalizations for disc herniation, which would be congruent with a genetic component to this condition. The classic twin studies reported to date also suggest a genetic
component to common spinal disorders and in some
cases, such as for disc degeneration, a substantial one that
overshadows the role of suspected environmental risk
factors. Specif ic gene forms associated with these conditions also have been identified, which may eventually
provide key insights into the mechanisms underlying
back disorders. Although the complex contributions and
interactions of genetic and environmental factors are currently unkno wn, these are fertile areas for future research.
REFERENCES
1. Riihimäki H. Low-back pain, its origin and risk indicators. Scand J
Work Environ Health 1991;17:81–90.
2. Frymoyer JW. Lumbar disk disease: epidemiology. Instr Course Lect
1992;41:217–223.
3. Ala-Kokko L. Genetic risk factors for lumbar disc disease. Ann Med
2002;34:42–47.
4. Loughlin J. Genetic epidemiology of primary osteoarthritis. Cur r Opin
Rheumatol 2001;13:111–116.
5. Khoury MJ, Beaty TH, Cohen BH. Fundamentals of genetic epidemiology. New York: Oxford University Press, 1993.
6. Cavalli-Sforza LL, Feldman MW Cultural transmissions and evolution:
a quantitative approach. Princeton, NJ: Princeton Univ ersity Press, 1981.
7. Sing CF, Moll PP. Genetics of atherosclerosis. Annu Rev Genet 1990;
24:171–187.
8. Solovieva S, Lohiniva J, Leino-Arjas P, et al. COL9A3 gene polymorphism and obesity in intervertebral disc degeneration of the lumbar
spine: evidence of gene-environment interaction. Spine 2002;27(23):
2691–2696.
9. Zebra KE, Sing CF. The role of genome type-environment interaction
and time in understanding the impact of genetic polymorphisms on
lipid metabolism. Curr Opin Lipidol 1993;4:152–162.
10. Cherkin DC, Deyo RA, Loeser JD, et al. An international comparison
of back surgery rates. Spine 1994;19:1201–1206.
11. Matsui H, Tsuji H, Terahata N. Juvenile lumbar herniated nucleus pulposus in monozygotic twins. Spine 1990;15:1228–1230.
12. Gunzburg R, Fraser RD, Fraser GA. Lumbar intervertebral disc prolapse in teenage twins. A case report and review of the literature. J
Bone Joint Surg Br 1990;72:914–916.
13. Matsui H, Terahata N, Tsuji H, et al. Familial predisposition and clustering for juvenile lumbar disc herniation. Spine 1992;17:1323–1328.
14. Varlotta GP, Brown MD, Kelsey JL, et al. Familial predisposition for
herniation of a lumbar disc in patients who are less than twenty-one
years old. J Bone Joint Surg Am 1991;73:124–128.
15. Nelson CL, Janecki CJ, Gildenberg PL, et al. Disk protrusions in the
young. Clin Orthop 1972;88:142–150.
16. Porter RW, Thorp L. Familial Aspects of disc protrusion. Orthop Trans
1986;10:524.
17. Scapinelli R. Lumbar disc herniation in eight siblings with a positive
family history for disc disease. Acta Orthop Belg 1993;59:371–376.
18. Varughese G, Quartey GR. Familial lumbar spinal stenosis with acute
disc herniations. Case reports of four brothers. J Neurosurg 1979;51:
234–236.
19. Wilde GP, Narth AD, Kerslake R. The familial incidence of disc degeneration. In: Proceedings of the Annual Meeting of the Society for Back
Pain Research, Oswestry, England. Hastings, England: Society for
Back Pain Research, 1990.
20. Postacchini F, Lami R, Pugliese O. Familial predisposition to discogenic low-back pain. An epidemiologic and immunogenetic study.
Spine 1988;13:1403–1406.
21. Simmons ED Jr, Guntupalli M, Kowalski JM, et al. Familial predisposition for degenerative disc disease. A case-control study. Spine 1996;
21:1527–1529.
22. Richardson JK, Chung T, Schultz JS, et al. A familial predisposition
toward lumbar disc injury. Spine 1997;22:1487–1492; discussion 1493.
23. Matsui H, Kanamori M, Ishihara H, et al. Familial predisposition for
lumbar degenerative disc disease. A case-control study. Spine 1998;23:
1029–1034.
24. Heikkilä JK, Koskenvuo M, Heliövaara M, et al. Genetic and environmental factors in sciatica. Evidence from a nationwide panel of 9365
adult twin pairs. Ann Med 1989;21:393–398.
25. Annunen S, Paassilta P, Lohiniva J, et al. An allele of COL9A2 associated with intervertebral disc disease. Science 1999;285:409–412.
26. Karppinen J, Pääkkä E, Raina S, et al. Magnetic resonance imaging
findings in relation to the COL9A2 tryptophan allele among patients
with sciatica. Spine 2002;27:78–83.
27. Paassilta P, Lohiniva J, Goring HH, et al. Identification of a novel common genetic risk factor for lumbar disk disease. JAMA 2001;285:
1843–1849.
28. Bull J, el Gammal T, Popham M. A possible genetic factor in cervical
spondylosis. Br J Radiol 1969;42:9–16.
29. King JB. A radiographic survey of 9 pairs of elderly identical twins.
Clin Radiol 1968;19:315–317.
30. King JB. A radiographic survey of nine pairs of elderly identical twins.
Clin Radiol 1971;22:375–378.
31. Hurxthal LM. Schmorl’s nodes in identical twins: their probable
genetic origin. Lahey Clin Foundation 1966;15:89–92.
106 /SECTION I/BASIC SCIENCE
32. Langinvainio H, Koskenvuo M, Kaprio J, et al. Finnish twins reared
apart. II: Validation of zygosity, environmental dissimilarity and
weight and height. Acta Genet Med Gemellol (Roma) 1984;33:
251–258.
33. Battie MC, Videman T, Gibbons LE, et al. 1995 Volvo Award in clinical sciences. Determinants of lumbar disc degeneration. A study relating lifetime exposures and magnetic resonance imaging findings in
identical twins. Spine 1995;20:2601–2612.
34. Battie MC, Haynor DR, Fisher LD, et al. Similarities in degenerative
findings on magnetic resonance images of the lumbar spines of identical twins. J Bone Joint Surg Am 1995;77:1662–1670.
35. Sambrook PN, MacGregor AJ, Spector TD. Genetic influences on cervical and lumbar disc degeneration: a magnetic resonance imaging
study in twins. Arthritis Rheum 1999;42:366–372.
36. Videman T, Leppävuori J, Kaprio J, et al. Intragenic polymorphisms of
the vitamin D receptor gene associated with intervertebral disc degeneration. Spine 1998;23:2477–2485.
37. Jones G, White C, Sambrook P, et al. Allelic variation in the vitamin D
receptor, lifestyle factors and lumbar spinal degenerative disease. Ann
Rheum Dis 1998;57:94–99.
38. Videman T, Gibbons LE, Battie MC, et al. The relative roles of intragenic polymorphisms of the vitamin d receptor gene in lumbar spine
degeneration and bone density. Spine 2001;26:E7–E12.
39. Kawaguchi Y, Osada R, Kanamori M, et al. Association between an
aggrecan gene polymorphism and lumbar disc degeneration. Spine
1999;24:2456–2460.
40. Takahashi M, Haro H, Wakabayashi Y, et al. The association of degeneration of the intervertebral disc with 5a/6a polymorphism in the promoter of the human matrix metalloproteinase-3 gene. J Bone Joint Surg
Br 2001;83:491–495.
41. Kimura T, Nakata K, Tsumaki N, et al. Progressive degeneration of
articular cartilage and intervertebral discs. An experimental study in
transgenic mice bearing a type IX collagen mutation. Int Orthop 1996;
20:177–181.
42. Palmer PE, Stadalnick R, Arnon S. The genetic factor in cervical
spondylosis. Skeletal Radiol 1984;11:178–182.
43. Bachiocco V, Scesi M, Morselli AM, et al. Individual pain history and
familial pain tolerance models: relationships to post-surgical pain. Clin
J Pain 1993;9:266–271.
44. Edwards PW, Zeichner A, Kuczmierczyk AR, et al. Familial pain models: the relationship between family history of pain and current pain
experience. Pain 1985;21:379–384.
45. Lester N, Lefebvre JC, Keefe FJ. Pain in young adults: I. Relationship
to gender and family pain history. Clin J Pain 1994;10:282–289.
46. Violon A, Giurgea D. Familial models for chronic pain. Pain 1984;18:
199–203.
47. MacGregor AJ, Griffiths GO, Baker J, et al. Determinants of pressure
pain threshold in adult twins: evidence that shared environmental influences predominate. Pain 1997;73:253–257.
48. Bengtsson B, Thorson J. Back pain: a study of twins. Acta Genet Med
Gemellol (Roma) 1991;40:83–90.
49. MacGregor AJ, Andrew T, Snieder H, et al. A genetic model for lower
back pain: a population-based MRI study of twins. Presented at the
annual meeting of the American College of Rheumatology, Session B:
poster 452,1999.
50. Mogil JS. The genetic mediation of individual differences in sensitivity
to pain and its inhibition. Proc Natl Acad Sci U S A 1999;96:7744–7751.
51. Gaertner RL. Idiopathic scoliosis in identical (monozygotic) twins.
South Med J 1979;72:231–234.
52. Robin GC, Cohen T. Familial scoliosis. A clinical report. J Bone Joint
Surg Br 1975;57:146–148.
53. Axenovich TI, Zaidman AM, Zorkoltseva IV, et al. Segregation analysis of idiopathic scoliosis: demonstration of a major gene effect. Am J
Med Genet 1999;86:389–394.
54. Axenovich TI, Zaidman AM, Zorkoltseva IV, et al. Segregation analysis of Scheuermann disease in ninety families from Siberia. Am J Med
Genet 2001;100:275–279.
55. Hayek S, Laplaza FJ, Axelrod FB, et al. Spinal deformity in familial
dysautonomia. Prevalence, and results of bracing. J Bone Joint Surg
Am 2000;82-A:1558–1562.
56. Haukipuro K, Keränen N, Koivisto E, et al. Familial occurrence of
lumbar spondylolysis and spondylolisthesis. Clin Genet 1978;13:
471–476.
57. Wynne-Davies R, Scott JH. Inheritance and spondylolisthesis: a radiographic family survey. J Bone Joint Surg Br 1979;61-B:301–305.
58. Giampietro PF, Raggio CL, Blank RD. Synteny-defined candidate
genes for congenital and idiopathic scoliosis. Am J Med Genet 1999;
83:164–177.
59. Wise CA, Barnes R, Gillum J, et al. Localization of susceptibility to
familial idiopathic scoliosis. Spine 2000;25:2372–2380.
60. Miller NH. Spine update. Genetics of familial idiopathic scoliosis.
Spine 2000;25:2416–2418.
61. Simper LB. Spondylolysis in Eskimo skeletons. Acta Orthop Scand
1986;57:78–80.
62. Mathiesen F, Simper LB, Seerup A. Multiple spondylolyses and
spondylolistheses. Br J Radiol 1984;57:338–340.
63. Al-Sebai MW, Al-Khawashki H. Spondyloptosis and multiple-level
spondylolysis. Eur Spine J 1999;8:75–77.
64. Ravichandran G. Multiple lumbar spondylolyses. Spine 1980;5:552–557.
65. Martin RP, Deane RH, Collett V. Spondylolysis in children who have
osteopetrosis. J Bone Joint Surg Am 1997;79:1685–1689.
66. Igarashi S, Koyama T, Shimosaka S, et al. [Familial narrow spinal canal
(lumbar canal stenosis with narrow cervical canal): case reports of
three brothers]. No Shinkei Gekakei Geka 1982;10:961–966.
67. Postacchini F, Massobrio M, Ferro L. Familial lumbar stenosis. Case
report of three siblings. J Bone Joint Surg Am 1985;67:321–323.
68. Iida H, Shikata J, Yamamuro T, et al. A pedigree of cervical stenosis,
brachydactyly, syndactyly, and hyperopia. Clin Orthop 1989:80–86.
69. Hughes PJ, Edwards JM, Ridler MA, et al. A balanced autosomal
translocation (3;9) associated with primary hypogonadism and dorsal
spine stenosis. Clin Genet 1993;43:44–45.
70. Hamanishi C, Nagata Y, Nagao Y, et al. Acrodysostosis associated with
spinal canal stenosis. Spine 1993;18:1922–1925.
71. Adams JE, Davies M. Intra-spinal new bone formation and spinal cord
compression in familial hypophosphataemic vitamin D resistant osteomalacia. Q J Med 1986;61:1117–1129.
CHAPTER 9
Genetic Applications to Lumbar Disc Disease
Christian Lattermann, Lars G. Gilbertson, and James D. Kang
The etiology and pathophysiology of degenerative disc
disease (DDD) are still unknown. However, it is believed,
that it is the result of a complex interaction between biologic and mechanical factors.
New biologic techniques may allow for addressing
intervertebral disc degeneration on a molecular level.
Recent advancements in recombinant DNA technology
have led to the decoding of many human genes that
appear to be attractive for the scientific and clinical use
in musculoskeletal disorders (1,2). Growth factors and
embryogenic differentiation factors have been isolated
and studied for many musculoskeletal conditions. Bone
morphogenetic proteins (BMPs), for example, are successfully being used to enhance bone healing and fusion
in humans (3). Other growth factors such as transforming
growth factor β (TGF-β), or insulin-derived growth factor 1 (IGF-1) have been shown to be able to influence the
proliferation and extracellular matrix production of various different musculoskeletal tissues (4–6).
After this brief overview we will introduce several
novel approaches involving molecular genetic techniques
and how their use can be advantageous for the treatment
of DDD in the lumbar spine.
IDENTIFICATION OF GENES FOR TARGETED
GENE MANIPULATION
Intervertebral disc disease occurs because of a complex interaction of cells, cell products, inflammatory
cytokines, and degradative processes occurring in the
intervertebral disc. All of these to-date identified mechanisms are naturally occurring processes that are designed
to maintain the intervertebral disc homeostasis. One or
multiple unknown triggers mark the beginning of disc
degeneration by causing a shift of the anabolic/catabolic
equilibrium. The goal of any biologic therapy for DDD
therefore must be to reinstate the equilibrium or slow
down the shift of the anabolic/catabolic equilibrium.
In order to be able to identify the different pathways in
which a gene therapy protocol would be able to intervene
toward a slowing of the degenerative process one has to
understand the process of disc degeneration. While there
are still many secrets to be solved in the comple x process
of disc degeneration it seems that there is a fundamental
concept of homeostasis that is gradually disrupted during
the degeneration of the intervertebral disc. To facilitate
the understanding of the complex process of intervertebral disc degeneration and the possible ways of therapeutic intervention one can group the different mechanisms
responsible for maintenance of disc homeostasis into two
major categories: nutritional and catabolic.
Nutritional
One of the first steps in disc degeneration may be the
increase in fibrochondrocytes along the annulus fibrosus.
This increased fibrosis has been observed parallel to a
decrease in diffusion of substances throughout the intervertebral disc. This in turn may be responsible for the
declining oxygen tension within the intervertebral disc. A
decrease in oxygen tension most likely will result in
impairment of cellular function within the nucleus and
thus may lead to a decrease in matrix synthesis.
Decreased matrix synthesis will lead to a favored production of the smaller, less complex keratan sulfate shifting the equilibrium toward a higher concentration of
nonaggregated proteoglycans that bind fewer water molecules (7–9). As a result, the overall capacity of the
nucleus pulposus to imbibe water decreases. In addition
there seems to be an abundance of smaller proteoglycan
fragments that appear in early disc degeneration secondary to the collapse of adequate matrix proteoglycan
production. These smaller, nonaggregate proteoglycans
and breakdown products decrease the fluid flow throughout the disc and thus, even further inhibit the diffusion
capacity of nutrients throughout the disc. This again lim-
107
108 /SECTION I/BASIC SCIENCE
its the oxygen tension and nutrient supply to and from the
intervertebral disc cells.
A further cascade involved in disruption of normal disc
homeostasis is the constant maintenance of different collagen types within the intervertebral discs. The intervertebral disc is predominantly composed of type I and II collagen. The annulus is predominantly composed of type I
collagen fibers. Type II collagen is mainly found in the
nucleus pulposus. The distribution shows a small gradient
toward the periphery, with the concentration of collagen
type II decreasing and type I collagen fibers increasing
toward the annulus. Despite the fact that this collagen
scaffold does not seem to change significantly during the
aging process, DDD shows significant alteration of the
collagen composition early on. In early de generation more
type I and II collagen is expressed, however, in tandem
with an increase in minor collagen types (III, V, VI). During the course of further degeneration collagen type II will
disappear in the nucleus and be replaced by collagen type
I. The minor collagen types of fibrosis (III, IV, and X)
become more abundant within the nucleus pulposus and
gradually lead to a loss of elasticity.
Catabolic
An inflammatory component has been discussed as a
major entity in degeneration of the intervertebral disc.
Nitric oxide (NO), interleukin-1 (IL-1) and 6 (IL-6), and
prostaglandin E
(PGE2) are pow erful inflammatory medi-
2
ators which have been shown to be elevated in degenerated human intervertebral discs. Although the mechanisms are not fully understood to date NO , IL-6, and PGE
appear to be up-regulated in response to the main inflammatory cytokine IL-1. It is likely that these inflammatory
mediators have multiple functions but one of their functions is to support the breakdown of proteoglycans mediated by degradative enzymes called matrix metalloproteinases (MMPs). These MMPs are a family of enzymes
responsible for the breakdown of collagens and extracellular matrix. The MMPs include well-known enzymes
such as, collagenase 1-3, gelatinases, stromelysin, or
aggrecanase. These powerful catabolic enzymes are able
to breakdown different sizes of matrix proteoglycans and
collagens and show a significantly higher activity in
degenerated intervertebral disc cells than in normal discs.
It is surprising, however, that the actual amount of MMPs
is not increased in the degenerated intervertebral disc. In
fact, the increase in proteoglycan breakdo wn may be more
likely a result of the lack of inhibition of the MMPs.
In a normal intervertebral disc MMPs are inhibited by
molecules called tissue inhibitors of metalloproteinases
(TIMPs). The concentration of these TIMPs is greatly
decreased in degenerated intervertebral discs. This mechanism, therefore, suggests a breakdown of the anti-catabolic system within the intervertebral disc during degeneration.
Strategies that result in a net increase in proteoglycans
may have therapeutic potential in altering the natural history of disc degeneration. These strategies could involve
increasing the production of proteoglycans, blocking
their catabolic degradation, or a combination of both.
Possible Targets for Gene or Protein Transfer
One common way to increase the producti vity of cells in
the presence of impaired function uses small proteins
called growth factors. These growth factors have the abil-
ity to override and steer cellular protein synthesis in less
than optimal surroundings. Naturally occurring, these
growth factors offer a way in which intervertebral disc
cells can be influenced and guided to produce extracellular
matrix and collagen when disc degeneration occurs and
thus counteract the degradation of the intervertebral disc.
Several promising growth factors have been isolated
which have the ability to increase extracellular matrix production and collagen production in intervertebral disc
cells. Transforming growth f actor-β (TGF-β1) and bone
morphogenetic proteins (BMPs) are two examples of
growth factors (out of many) with strong potential for
altering intervertebral disc (IVD) biology. Thompson et al.
studied the in vitro response of canine IVD tissue to the
following growth factors: human recombinant IGF-1, epidermal growth factor (EGF), fibroblast growth factor
(FGF), and TGF-β1. Incorporation rates by the tissue
regions of up to five times the control rate were reported,
with the nucleus and transition zone responding more than
the annulus. TGF-β1 and EGF elicited greater response
than FGF, while IGF-1 produced only a marginally signif-
2
icant response in the nucleus and no response in the annulus and transition zone (10). Our group showed that the use
of TGF-β1 and BMP-2 lead to higher levels of proteoglycan production in degenerative human and rabbit nucleus
pulposus cells (6). Takegami et al. studied the effect of
human recombinant osteogenic protein (hrOP-1) on cell
proliferation as well as on proteoglycan production and
collagen synthesis. They showed that there is a dosedependent increase in proliferation rate as well as collagen
and proteoglycan production of rabbit intervertebral disc
cells treated with hrOP-1. They were also able to show the
restoration of proteoglycan in previously proteoglycandepleted cultures of intervertebral disc cells if they were
treated with hrOP-1 (11). Our laboratory has just recently
shown that the treatment of degenerative intervertebral
disc cells with TIMP-1 will increase proteoglycan production and the rate of proteoglycan synthesis b y a factor of 5.
However, the critically important issue for the delivery
of growth factors is the length of therapeutic effect of
these exogenous growth factors to targeted cells in the
IVD. The normal half-life for most of these growth factors in vivo is approximately 20 minutes. Therefore, the
therapeutic effect of injecting growth factors directly into
the IVD may be too transient to ha v e a major long-lasting
CHAPTER 9/GENETIC APPLICATIONS TO LUMBAR DISC DISEASE / 109
effect on a chronic disorder such as DDD, and repeated
injections may not be practical or well tolerated by
patients.
THE CONCEPT OF GENE TRANSFER
A sophisticated way to deliver sustained levels of
growth factors to musculoskeletal tissues has been shown
to be gene transfer technology. Particularly, the use of
viral vectors appears to be highly ef ficient in the delivery
of the desired transgene to most mesenchymal tissues.
Gene transfer is a novel technique in which genes of
interest are inserted into target cells, causing them to synthesize the protein encoded by the inserted gene. This technique can be used as an approach for treating genetic diseases by compensating for mutant genes or as a means of
delivering a therapeutic substance to the area of interest.
Protein synthesis within a mammalian cell involv es several steps. At first a gene, consisting of specific DNA
sequence is transcribed into a complementary chain heterogeneous to nuclear RNA. This is then processed into
messenger RNA (mRN A) by a series of modifications that
include capping, splicing, and the addition of a polyadenosine tail. The mature mRNA leaves the nucleus of the cell
and is translated by ribosomes into a sequence of amino
acids that form the protein. When an exogenous gene is
introduced into the nucleus of a cell, it is also transcribed
into mRNA and thus produces the protein encoded by the
gene. The cell ma y normall y not make this protein of interest, or it may be made in insufficient amounts. There are
different techniques av ailab le that aid the insertion of a foreign gene into the genome of a mammalian cell. Gene
transfer to cells normally requires the assistance of a vehicle or vector, which may be viral or nonviral in nature. The
nonviral techniques typically use small particles like liposomes or spheroblasts carrying the gene of interest. These
particles have the ability to fuse with the target cell or to
enter the cell by endocytosis. Other techniques like elec-
troporation and microparticle bombardment use physical
strain or electric shock in order to break small temporary
defects into the cell wall without severely damaging the
cell, allowing the DNA strand to travel into the cell.
Another approach uses a direct microinjection of the gene
into the cell. These methods tend to be inefficient (5).
Viral-based vectors generally use the inherent capacity
of a virus to attach to the surface of a cell, through specific
receptors, and insert its genome into the cell (Fig. 9-1). For
safety reasons the viral vector must be altered to render the
virus incapable of replicating. Hence viral vectors are engineered such that endogenous gene sequences required for
replication and pathology are remo ved. The ideal viral vector therefore carries the genes of interest into cells with
high efficiency, but does not replicate or cause pathology.
By far the most commonly used vectors are retroviral
and most are based upon the Molony murine leukemia
virus. Retroviral vectors specifically infect dividing cells
with a very high efficiency. They insert their genes into
the chromosomes of the target cell. This leads to reproduction of the inserted gene each time the infected cell
divides. Clinical trials have already been successfully initiated using retroviral vectors. Although retroviral vectors
are the most commonly used vectors in human clinical trials at present, there are certain disadvantages in their use.
For example, retroviruses do not infect nondividing cells.
Furthermore, there is a theoretical risk of mutagenesis due
to the random integration of the viral DNA into the chromosome of the target cell. If chromosomal integration
occurs near a site of an oncogene, activation may occur
causing the cell to transform. Because of this potential
risk, most investigators have used the retroviral vectors in
an ex vivo approach (discussed later in the chapter).
Presently, however, there are no reports of malignancy
caused by gene therapy using retroviral vectors.
The second most commonly used viral vector is
derived from the adenovirus. This is a DNA virus that is
highly infectious to a number of different cell types. The
FIG. 9-1. The DNA coding for a growth factor is
engineered into a vector (i.e., adenoviral vector
capsid). The vector is applied to the tissue or cell
culture and attaches to the cell membrane. The
DNA is inserted into the cell and travels to the
nucleus where it integrates episomally or integrates into the chromosome. The inserted DNA
then uses the regular transcription and translation
process of the host cell and is translated into the
protein of interest. The treated cell now begins to
produce the protein of interest in high amounts.
110 /SECTION I/BASIC SCIENCE
adenoviral vector infects dividing as well as nondividing
cells and can be prepared in high titers. In contrast to the
retroviral vectors, the genome of the adenoviral vector is
not integrated into the chromosome of the target cell. The
adenoviral vector inserts its genome as an episome within
the nucleus of the target cell. Thus, the inserted genes will
not be automatically passed on during cell division. As a
result the percentage of infected daughter cells will
rapidly decrease as a result of dilution with every cell
generation. However, adenoviral vectors are highly antigenic and initiate strong immune responses. Likewise, herpes viral vectors, which have the capability of including
multiple transgenes, are also antigenic and often cytotoxic
to the host cell or tissues. Currently, new generations of
viral-based vectors are under development. These will
increase the efficiency of transduction of both dividing and
nondividing cells. The most promising of these are based
upon adeno-associated virus (AAV), the herpes simplex
virus (HSV), and the lenti-retrovirus. These v ectors sho w a
high infectivity and may provide a long-term expression.
The goal is to generate new viral vectors which can escape
the surveillance of the host immune system and which can
express the desired gene product in a tissue-specific manner.
Gene transfer can be accomplished by two main
approaches, ex vivo or in vivo, in order to transduce tar-
get cells.
The ex vivo approach transduces target cells after har-
vest and culture in vitro under sterile conditions. The cells
are transduced and selected in culture and then prepared
for injection into the recipient tissue. Because no viral
particles enter the human body, ex vivo gene therapy provides a measure of safety that is not found with in vivo
gene delivery.
In vivo transduction is a more straightforward procedure. The v ector is directly applied into the tissue of interest by catheter or needle injection. This approach however, does not allow control over the rate of target cell
transduction. Due to the direct introduction of viral particles into the body, safety concerns are higher. The choice
of the approach to achieve target cell transduction is
dependent upon the desired longevity of gene expression,
the viral vector chosen, the anatomy and physiology of
the target organ, safety considerations, and the underlying
cause of the disease to be treated. Generally, the ex vivo
approach is usually employed when using retroviral vectors because of the necessity for high rates of cell division
and safety concerns surrounding the injection of retrovirus into the body . Due to their high infectivity and ability to infect nondividing cells, adenoviral vectors are
often used experimentally in the in vivo approach (5).
Gene Transfer to the Intervertebral Disc
Several authors have previously shown successful
transfer of exogenous genes to musculoskeletal tissues. In
our laboratory we have pioneered the viral gene transfer
to the intervertebral disc using adenoviral and retroviral
gene transfer protocols.
Wehling et al. reported the successful gene transfer of
the LacZ marker gene as well as the interleukin receptor
antagonist gene (IRAP) to bovine intervertebral disc cells
(12). Subsequently, Nishida et al. performed a study
which showed that the adenoviral transfer of the LacZ
marker gene to the rabbit intervertebral disc is feasible
and will lead to long-term expression of the marker gene
(13). This has since been proved to be the case with different viral vectors including AAV (Figs. 9-2 and 93A,B). Surprisingly the intervertebral disc allowed for
long-term gene expression after use of an adenoviral vector, suggesting that the intervertebral disc may be an
immune-privileged site within the human body. This
observation has since been underlined by Park et al. He
found an unusually high expression of FAS ligand, a suppressor of cellular immunity, within the intervertebral
disc (14). In a follow-up study Nishida et al. transferred
the gene for TGF-β to rabbit intervertebral discs in vivo
and could show that the overall proteoglycan production
of the intervertebral disc cells increased (15). Moon et al.
FIG. 9-2. In vitro transduction of human intervertebral disc
cells with two different viral vectors. The adeno-associated
virus vector (AAV) and the adenoviral vector (ad) both trans-
fer the LacZ marker gene to human intervertebral disc cells.
Both viruses show a clearly dose-dependent transduction
efficacy. The adenoviral vector is overall more efficient. Adenoviral vectors have the advantage of efficient transduction
of nondividing cells. The AAV shares this advantage but in
addition is much less immunogenic and is not associated
with any known disease in humans. Thus, the AAV vectors
may be potentially safer than adenoviral or retroviral vectors.
CHAPTER 9/GENETIC APPLICATIONS TO LUMBAR DISC DISEASE / 111
A B
FIG. 9-3. In vivo transduction of intervertebral disc cells in rabbits can be achie v ed using a simple injec-
tion technique. Adenoviral gene transfer of the LacZ marker gene (A) at 6 weeks can be traced as long
as 1 year post-injection. The in vivo delivery of the LacZ marker gene using the novel AAV vector (B)
can be detected for at least 6 weeks post-injection.
investigated the effect of different growth factors transferred to human intervertebral disc cells using an adenoviral vector. He showed synergism between the expression of TGF-β, BMP-2, and IGF-1 with respect to the
overall proteoglycan production in culture (6). In a
recently published study Yung et al. applied a pellet culture technique in order to grow intervertebral disc cells in
a three-dimensional matrix. Transduction of these pellet
cultures with an adenoviral vector coding for the BMP-2
gene led to an increase in proteoglycan synthesis and total
proteoglycan content (16).
FUTURE PERSPECTIVES
Clearly there are still many obstacles to overcome
before a viral or nonviral gene transfer protocol can be
used as a viable treatment option in DDD. Molecular
biologists and surgeons, however, are feverishly working
to develop safer methods of gene transfer in order to be
able to influence the biologic environment within soft tissues such as the intervertebral disc. We know from animal experimental data that the approach is feasible in
vivo. Safety studies are currently underway to determine
if these technologies may be applicable to humans.
In addition to the development of novel and safe vectors researchers are developing new models to mimic
intervertebral discs in vitro and in vivo.
Finally, it is impor tant to understand the goal of any
therapeutic approach to disc degeneration. The major
issue to overcome at this time is still the early detection
of disc degeneration. Questions that need to be answered,
address the time course of degeneration. When is a disc
too degenerated for therapy? How much regeneration
potential does a degenerated disc have? How well does
the magnetic resonance image signal change correlate
with the biologic activity of the intervertebral disc? All
these questions will have to be addressed before a broad-
based attempt to treat this disease using gene therapy can
be made. As of now, we still do not know the exact cause
of disc degeneration. It is certainly not feasible to prophylactically treat all degenerated discs at all levels with
a gene therapy approach. It is therefore important to
focus treatment using this new technology to v ery limited
and clearly defined problems. One of these problems, for
example, is disc degeneration occurring above and below
fusions in the lumbar or cervical spine. At the time of
fusion an injection into the adjacent discs could be performed without any problems.
In conclusion, gene transfer technology offers a sophisticated way to influence the biochemical environment
inside the degenerated intervertebral disc and may be a
useful tool to treat this highly prevalent disease in the
future. The transfer of gro wth factors to the intervertebral
disc may be able to limit disc degeneration or it may be
able to prevent disc degeneration if the gene transfer is
done prophylactically at a junctional level at the time of
posterior spinal fusion. Viral or nonviral gene transfer is
an emerging technology that will be able to offer exciting
new perspectives in research and treatment of interver tebral disc disease.
REFERENCES
1. Evans CH, Scull y SP. Orthopaedic gene therapy. Clin Orthop 2000;379
[Suppl]:S2
2. Jaffurs D, Evans CH. The Human Genome Project: implications for the
treatment of musculoskeletal disease. J Am Acad Orthop Surg 1998;6
(1):1–14.
3. Boden SD, Zdeblick TA, Sandhu HS, et al. The use of rhBMP-2 in
interbody fusion cages. Definitive evidence of osteoinduction in
humans: a preliminary report. Spine 2000;25(3):376–381.
4. Puolakkainen PA, Twardzik DR, Ranchalis JE, et al. The enhancement in
wound healing by transforming growth factor-beta 1 (TGF-beta 1)
depends on the topical delivery system. J Surg Res 1995;58(3):321–329.
5. Gruber HE, Hanley EN Jr. Human disc cells in monolayer vs 3D culture: cell shape, division and matrix formation. BMC Musculoskeletal
Disord 2000;1(1):1.
6. Moon SH, Gilbertson LG, Nishida K, et al. Human intervertebral disc
112 /SECTION I/BASIC SCIENCE
cells are genetically modifiable by adenovirus-mediated gene transfer:
implications for the clinical management of intervertebral disc disorders. Spine 2000;15;25 (20):2573–2579.
7. Lipson SJ, Muir H. 1980 Volvo award in basic science. Proteoglycans in
experimental intervertebral disc degeneration. Spine 1981;6(3):194–210.
8. Pearce RH, Grimmer BJ, Adams ME. Degeneration and the chemical
composition of the human lumbar intervertebral disc. J Orthop Res
1987;5(2):198–205.
9. Stevens RL, Ryvar R, Robertson WR, et al. Biological changes in the
annulus fibrosus in patients with low-back pain. Spine 1982;7(3):
223–233.
10. Thompson JP, Oegema TR Jr, Bradford DS. Stimulation of mature
canine intervertebral disc by growth factors. Spine 1991;16(3):253–260.
11. Takegami K, Thonar EJ, An HS, et al. Osteogenic protein-1 enhances
matrix replenishment by intervertebral disc cells previously exposed to
interleukin-1. Spine 2002;15;27 (12):1318–1325.
12. Wehling P, Schulitz KP, Robbins PD, et al. Transfer of genes to chondrocytic cells of the lumbar spine. Proposal for a treatment strategy of spinal
disorders by local gene therapy. Spine 1997;15;22 (10):1092–1097.
13. Nishida K, Kang JD, Suh JK, et al. Adenovirus-mediated gene transfer
to nucleus pulposus cells. Implications for the treatment of intervertebral disc degeneration. Spine 1998;15;23 (22):2437–2442.
14. Park JB, Chang H, Kim KW. Expression of Fas ligand and apoptosis of
disc cells in herniated lumbar disc tissue. Spine 2001;15;26 (6):618–621.
15. Nishida K, Kang JD, Gilbertson LG, et al. Modulation of the biologic
activity of the rabbit intervertebral disc by gene therapy: an in vivo
study of adenovirus-mediated transfer of the human transforming
growth factor beta 1 encoding gene. Spine 1999;1;24 (23):2419–2425.
16. Yung Lee J, Hall R, P elink ovic D, et al. New use of a three-dimensional
pellet culture system for human intervertebral disc cells: initial characterization and potential use for tissue engineering. Spine 2001;1;26
(21):2316–2322.