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ACTA OPTOP BPAS 15(1) - 2007

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ORIGINAL ARTICLE
FRANCINE TERESA BRIONI NUNES
1
, NIVEA DULCE TEDESCHI CONFORTI-FROES
2
, WILSON FBIO NEGRELLI
3
, DOROTIA ROSSI SILVA SOUZA
4
SUMMARY
7|e e||c|c, c/ |n|e|.e||eo|a| o|sc oeene|a||cn (|DD; |as nc| oeen
/0||, c|a||/|eo ,e|. v||am|n D |eceo|c|s ene (vDP; |as oeen s0es|-
eo as cne c/ ||e oc|en||a| en||||es |n.c|.eo |n o|sc oa||c|c|es cnse|.
On ||e c||e| |ano, |||s s|0o, cc||e|a|es, /c| ||e /||s| ||me, |0|a|||cne
||ans/e|ases V' ano 7' enes (GS77' ano GS7V'; oa|||c|oa||cn,
.||c| a|e |esocns|o|e /c| c|a|e||e ccmocnen|s |nac||.a||cn, |n |DD.
DN/ .as e||ac|eo /|cm |e0|cc,|es c/ oa||en|s ano 88 ccn||c|s,
oa||eo o, enoe| ano ae. 7|e vDP-Fc|| oc|,mc|o||sm .as am-
o||/|eo o, oc|,me|ase c|a|n |eac||cn (|CP; /c||c.eo o, |es|||c||cn
.||| Fc|| enz,me. GS77'/V' oc|,mc|o||sms .e|e oe|e|m|neo o,
means c/ |CP m0|||o|e. Fam||, ||s|c|, ano o|sease se.e|||, .e|e
|||||||eo |n oa||en|s ca||,|n ||e / a||e|e c/ ||e vDP-Fc|| ene
(|~0.000 ano 0.00'2, |esoec||.e|,;. 7|e ae a| o|sease cnse| |as
s|c.n |c oe ea||, |n |no|.|o0a|s .||| _// enc|,oe (a.e|ae 2 ,ea|s
c|o;. / cc||e|a||cn .as /c0no oe|.een Fc|| oc|,mc|o||sm ano ea||,
oeene|a||cn ano |DD se.e|||,, .||| smc||n |ao|| a|sc |n|e|/e||n |n
|||s o|ccess, |ea|o|ess c/ ||e o|esence c| aosence c/ a /a.c|ao|e
enc|,oe /c| GS77'/V'.
Keywords: P|s| /ac|c|s, |c|,mc|o||sm, ene||c, |n|e|.e||eo|a|
o|sc.
C/tat/on: N0nes F7B, Con/o|||-F|oes ND7, Neg|e||| wF, So0za DPS. Gene||c and ent||onmen|a| /ac|o|s |nto|ted on |n|e|te||eo|a| d|sc degene|a||on. 4c|a O||op
B|as. [se||a| on ||e /n|e|ne|j. 2007; 15(1}. 09-13. 4ta||ao|e /|om UP/. |||p.//www.sc|e|o.o|/aoo.
INTRODUCTION
The |ntervertebra| d|sc |oses |ts hygroscop|c power w|th age, caus|ng
a progress|ve dehydrat|on process, character|z|ng a d|scopathy. From
|ntervertebra| d|sc degenerat|on, the sp|ne beg|ns to show a progress|ve
|nstab|||ty on the |nvo|ved area, |ead|ng to a number o urther events on
the other e|ements o the segmenta| unct|ona| un|t. Th|s may occur on
any reg|on o the sp|ne, however, |t |s more requent|y ound between
the ourth and |th |umbar vertebrae, and between the |th |umbar and
|rst sacra|, at the age o th|rty orty years approx|mate|y
(1)
.
The prec|se et|o|ogy o lDD has not been u||y exp|a|ned. nt|| recent-
|y, |t was attr|buted un|que|y to the accumu|at|on o env|ronmenta|
eects, pr|mar||y m|cro or macro, trauma, ||esty|e, smok|ng hab|t,
atherosc|eros|s, added by changes occurr|ng on d|scs structure
w|th age. Neverthe|ess, more recent researches show a moderate
|n|uence o these actors on d|sc degenerat|on, wh|ch re|norces
the re|evance o a genet|c-re|ated actor or th|s d|sease
(2,3)
. Desp|te
o that, the natura| evo|ut|on o the degenerat|on wh|ch |s genet|-
ca||y determ|ned may be mod||ed to a certa|n extent by chang|ng
||esty|es and the env|ronment
(4)
.
I. PoIymorphism of Vitamin-D receptor gene on disc degen-
eration
The act|ve orm o v|tam|n-D, the 1_,25 d|-hydroxy-v|tam|n D [1_,25-
(OH)2D3] or v|tam|n-D3, |s necessary or ce|| growth and d|eren-
t|at|on on many t|ssues. Furthermore, |t |s |nvo|ved on bone m|ner-
a||zat|on process, ca|c|um absorpt|on on the |ntest|ne, phosphorus
homeostas|s, and parathyro|da| hormone regu|at|on. Add|t|ona||y,
|t part|c|pates on the d|erent|at|on, pro||erat|on and maturat|on o
cart||ag|nous ce||s, a|so |n|uenc|ng proteog|ycans synthes|s through
|o|nt chondrocytes
(5)
.
The v|tam|n-D receptor (vDP) |s a member o the super am||y o hor-
mona| nuc|ear receptors regu|at|ng the transcr|pt|on. Some o these
members must be h|gh||ghted, |nc|ud|ng: the stero|d and ret|no|c ac|d
receptors (PXP), wh|ch have a stero|d-bond|ng doma|n, an |nteract|on
doma|n w|th receptors' 'super am||y' members, and a th|rd DNA-
GENETIC AND ENVIRONMENTAL FACTORS INVOLVED
ON INTERVERTEBRAL DISC DEGENERATION
Study conducted at the Mo/ecu/ar E/dem/o/ogy Laboratory - Pau//sta State Un/vers/ty -UNESP, So Jos do R/o Preto. B/ochem/stry and Mo/ecu/ar B/o/ogy Laboratory, Med/ca/ Schoo/,
So Jos do R/o Preto (FAMERP)
Corresondences to: Nvea Du/ce Tedesch/ Confort/-Froes. Rua Mar/a F/gue/redo, 343/ 51- Paraso - CEP 04002-000 So Pau/o- Ca/ta/ - E-ma//: nfroes@/b//ce.unes.br
1. Master /n Genet/cs, Pau//sta State Un/vers/ty, UNESP.
2. Master and PhD /n Genet/cs, Pau//sta State Un/vers/ty, UNESP; ost-PhD, Un/vers/ty of Texas, Prevent/ve Med/c/ne Deartment, Ga/veston, USA.
3. Orthoaed/c and Traumato/ogy doctor.
4. Master, Un/vers/ty of So Pau/o (USP) and PhD /n Genet/cs, Pau//sta State Un/vers/ty, UNESP.
Pece|ved |n: 04/12/06, approved |n: 09/28/06
bond|ng doma|n, wh|ch conta|n two z|ng |ngers reasons, |ocated on
prote|n's N-end port|on
(6)
.
v|tam|n D3 [1_,25-(OH)2D3] |s ||poso|ub|e and, when enter|ng |nto
the ce||, |t prompt|y |nteracts w|th |ts receptor (vDP). Ater the vDP |s
act|vated by gett|ng bonded to v|tam|n D3, th|s receptor may, e|ther
separate|y or as a heterod|mer w|th severa| other super am||y's recep-
tors, part|cu|ar|y PXP, be bonded to the spec||c regu|at|on sequence,
present on the promoters o genes respons|ve to v|tam|n D. Fo||ow|ng
the heterod|mer ormat|on, transcr|pt|ona| actor llB (TFllB) and |ts co-
actors are recru|ted, thus start|ng transcr|pt|on
(7)
.
The Fokl po|ymorph|sm o the vDP gene resu|ts rom a change on
transcr|pt|on |n|t|a| s|te, |ocated at exon 2. That s|te, h|gh|y preserved
|n m|ce, rats and human be|ngs, suggests that the po|ymorph|sm on
vDP's exon 2 cou|d aect vDP prote|n unct|on|ng
(8)
.
II. EnvironmentaI Factors on IntervertebraI Disc Degenera-
tion
Smok|ng hab|t |nduces b|ochem|ca| stress to severa| t|ssues. Such
stress |s part o the env|ronmenta| components contr|but|ng to nutr|-
t|on and b|ood |ow changes on the d|sc, promot|ng a d|scogen|c
d|sease. There a|so are two add|t|ona| k|nds o stress, the dynam|c
and the phys|o|og|ca| one. The dynam|c stress |nvo|ves heavy act|v|-
t|es, overwe|ght, posture errors, and acc|denta| |n|ur|es, wh||e the
phys|o|og|ca| one |s assoc|ated to the ag|ng process
(9)
.
Among the most tox|c chem|ca| components o tobacco, n|cot|ne,
benzpyrene, and other po|ycyc||c aromat|c hydrocarbons are
ound. The |atter |ead to endothe||a| |n|ur|es, wh||e carbon monox-
|de, resu|t|ng rom combust|on, reduces red b|ood ce||s' ab|||ty to
transport oxygen. Catecho|am|ne |ncrease a|so occurs, resu|t|ng |n
vasoconstr|ct|on, urther ||m|t|ng oxygen supp|y to t|ssues and caus-
|ng damages to vertebrae and bones. There are ep|dem|o|og|ca|
ev|dences show|ng that chron|c smokers suer rom ear|y degen-
erat|on o the |ntervertebra| d|sc, hav|ng |ess res|||ent vertebrae, |n
add|t|on o present|ng bone dem|nera||zat|on advancement, wh|ch
may hasten osteoporos|s deve|opment
(10)
.
ACTA OPTOP BPAS 15(1) - 2007
10
occupat|ona| act|v|t|es, k|nd o d|et, hab|ts, and prov|ded the|r
anthropometr|c measurements or ca|cu|at|ng the body mass |ndex
(BMl) [we|ght (Kg)/ he|ght (m)].
II. 1. Patients' Assessment
Pat|ents' assessment was perormed by an expert doctor |n sp|na|
d|seases, w|th d|agnos|s be|ng corroborated by another qua|||ed
|nvest|gator. The ana|ys|s cons|sted o anamnes|s, am|||a| h|story
or sp|na| d|seases, phys|ca| exam|nat|on, and MP |mag|ng stud|es
o the sp|ne. From the MP, some data were extracted such as the
presence o d|scopathy and hern|at|on at d|erent degrees (convex|ty,
protus|on, extrus|on), s|gn |ntens|ty, he|ght, |brous r|ng and pu|pous
nuc|eus rupture, arthr|t|c |n|ur|es and ||gament degradat|on. Hern|at|on
|s understood as the |ntervertebra| d|sc protrud|ng out o |ts space to
the vertebra| body, s|gn |ntens|ty degree as a change on d|sc co|or,
rang|ng rom ||ght gray to b|ack, w|th darken|ng be|ng assoc|ated to
a dehydrat|on or degenerat|on process, he|ght as the degenerat|ve
process parameter, w|th |ts reduct|on be|ng assoc|ated to p|cture
evo|ut|on, |brous r|ng and pu|pous nuc|eus rupture as a cont|nu|ty
gap on |ts t|ssues, and, arthr|t|c |n|ur|es as |o|nts degenerat|on. F|gures
1 and 2 show degenerat|on s|gns assessed on pat|ents.
The d|sease sever|ty degree was |dent||ed accord|ng to the mod||ed
Schene|der's c|ass||cat|on
(5)
. ln th|s case, Degree 1 was character-
|zed by the presence o 1-3 s|gns above (no pat|ent showed |ess than
2 s|gns), Degree 2, w|th 4-5 s|gns, and Degree 3 show|ng 6-7 s|gns
o d|sc degenerat|on. S|m||ar|y, sever|ty |eve|s were attr|buted to the
d|sease, w|th |eve|s 1 and 2, respect|ve|y, emp|oyed |n cases o |n|ury
present |n one or more sp|na| segments.
II. 2. Genotyping
ln each sub|ect, 5mL o per|phera| b|ood were co||ected. The DNA
was obta|ned by us|ng the extract|on techn|que as descr|bed
by Gust|nc|ch e| a|.
(13)
.
The amp|||cat|on o the po|ymorph|c segment o the vDP-Fc|| gene
occurred by po|ymerase cha|n react|on (PCP). The detect|on o the
Fc|| po|ymorph|sm o||owed the protoco| descr|bed by Harr|s et a|.
(14)
.
The o||me|s: 5'-AGCTGGCCC TGGCACTGACTCTGCTCT-3' and 5'-
ATGGAAACACCTTGCTTCTTCTCCCTC-3' were d||uted |nto 1.5 mM
magnes|um ch|or|de, 60 mM Tr|s HC|, pH 9.0, 15 mM NH
4
SO
4
10%
dymethy|su|ox|de (DMSO), dNTPs (200[um]M each), 0.250L Taq po|y-
merase and genom|c DNA at 200ng/500L strength. The amp|||cat|on
cond|t|ons were as o||ows: 94`C or 30 seconds or DNA denaturat|on,
r|ng|ng temperature o 60`C or 30 seconds, 72`C or 30 seconds dur-
|ng 35 extens|on cyc|es. Generated byproducts were d|gested by Fc||
enzyme at 37`C or 3 hours, and then subm|tted to e|ectrophores|s |n
2% agarose ge|, conta|n|ng Tr|s-EDTA tampon and eth|d|um brom|de
(10mg/mL). The homozygous genotype named FF, does not conta|n
the restr|ct|on s|te or Fok| enzyme, thus resu|t|ng |n a u|| segment o
265 pb. The presence o the restr|ct|on s|te or th|s enzyme resu|ts |n a
ragment o 196 pb and a 69 pb e|se. ln these cond|t|ons, the homozy-
gous genotype |s categor|zed as .
Thus, the heterozygous genotype
F conta|n three bands, one o 265
pb, other o 196pb, and the |ast
one o 69 pb.
The po|ymorph|sms o the genes
GS77' and GS7V' were de-
tected by gen|c amp|||cat|on
by means o po|ymerase cha|n
react|on (PCP Mu|t|p|ex) de-
scr|bed by Abde|-Pahman et
a|.
(15)
. For PCP procedure, 30
pmo| o each o the o||me|s or
genes GS7V' (5'-GAACTCCCT-
GAAAAGCTAAAGC, 5'-GTT-
GGGCTCAAATATACGGTGG),
GS77' (5'-TTCCTTACTGGTCCT-
CACATCTC, 5'-TCACCGGAT-
CATGGCCAGCA) and CY|'/'
F/ gure 1 - N0c| ea| magne|| c
|esonance |mage, a| sag|||a| p|ane,
s|ow|ng d|scopa|||es a| /3-/4, /4-
/5 e /5-S1 |ete|s (o|ac| d|scs}.
Chem|ca| compounds present |n c|garettes are b|ometabo||zed,
be|ng ox|d|zed on phase l, ma|n|y, by the enzymes be|ong|ng to
cytochrome P-450 (CYPs)' super am||y, and |nact|vated on phase ll
by the con|ugat|on o the unct|ona| group o products ormed w|th
a hydro so|ub|e endogenous byproduct (g|utath|one, su|ate, g|u-
cose, acetate) by means o the g|utath|one-S-transerases (GSTs),
DP-g|ucorony|transerases and N-acety|transerases (NATs). Thus,
metabo||tes are transormed |nto hydroph|||c compounds, more
eas||y excreted
(11)
.
lnd|v|dua| d|erences on the ab|||ty to respond to genotox|c and
carc|nogen|c compounds are bonded to the presence o po|y-
morph|sms on enzymes |nvo|ved on the metabo||sm o chem|ca|
compounds, w|th th|s |nter-|nd|v|dua| var|ab|||ty p|ay|ng a ma|or ro|e
|n d|seases deve|opment.
Among phase-ll enzymes, GSTs p|ay a ma|or ro|e on detox||cat|on
processes. The gene GS77', |ocated on human's chromosome
22, |s po|ymorph|c. lts homozygous de|et|on may occur, resu|t|ng
|n a nu|| genotype (GS77' 0/0;, o wh|ch requency depends on
ethn|ca| or|g|n. The gene cod|y|ng to |soorm GS7V' |s |ocated at
chromosome 1, |s po|ymorph|c, present|ng our a||e||c var|ants, the
GS7V'*/, *B, *C, and *0. The |rst two are not d|erent regard|ng
the k|nd o byproduct, wh||e the a||e|e *C |s extreme|y rare and the
var|ant *0, wh|ch |s the nu|| a||e|e, causes absence o enzymat|c
act|v|ty when |n homozygos|s
(12)
. Thus, the presence o at |east
one unct|ona| a||e|e |s enough or the enzyme to unct|on by p|ay-
|ng |ts ro|e.
ln br|e, the smok|ng hab|t seems to be |nvo|ved |n |ntervertebra|
d|sc degenerat|on as an |mportant r|sk actor or th|s d|sease |n
human be|ngs.
OBJECTIVES
The purposes o th|s study |nc|ude the o||ow|ng:
1. ldent|y po|ymorph|sms on the v|tam|n-D receptor gene (vDP)
and the cod|y|ng gene or g|utath|one S-transerases M1 and T1
(GSTM1 and GSTT1) |n a group o pat|ents w|th |ntervertebra| d|sc
degenerat|on, as we|| as |n a contro| group,
2. Assoc|ate the po|ymorph|sms descr|bed above w|th the |nd|ngs
obta|ned rom nuc|ear magnet|c resonance, c||n|ca| data, am|||a| h|s-
tory and age at the onset o the d|sease, as we|| as anthropometr|c
(overwe|ght) and env|ronmenta| (smok|ng hab|t) actors,
3. Corre|ate anthropometr|c, occupat|ona|, env|ronmenta| and
am|||a| h|story actors or d|scogen|c d|sease between pat|ents
and contro|s.
MATERIALS AND METHODS
I. Case series
S|xty s|x pat|ents were stud|ed (38 ma|es and 28 ema|es), not re|ated
and screened |n a sp|ne-spec|a||zed c||n|c |n So Pau|o c|ty. A|| pa-
t|ents were Caucas|ans, ages rang|ng rom 16 to 62 years (average:
38 years o|d), present|ng w|th c||n|ca| and Magnet|c Pesonance (MP)
|mag|ng d|agnos|s o d|scopath|es, w|th or w|thout assoc|ated d|sc
hern|as. lnd|v|dua|s w|th d|sc changes proven to be resu|tant rom
prev|ous trauma, h|story o |obs requ|r|ng carry|ng heavy |oads,
excess|ve eorts, use o v|brat|ng mach|nes, or perorm|ng |mpact
act|v|t|es were exc|uded rom the study. The pat|ents were pa|red
to 88 Caucas|an sub|ects, ages rang|ng rom 16 to 57 years (aver-
age: 41 years o|d), be|ng 35 ma|es and 53 ema|es, screened at the
Med|ca| Co||ege |n So Jose do P|o Preto (FAMEPP) and at Pau||sta
State n|vers|ty "J||o de Mesqu|ta F||ho", campus So Jose do P|o
Preto, c||n|ca||y assessed and d|agnosed as d|scopath|es-ree. A||
sub|ects enro||ed have been |normed about the ob|ect|ves o the
study, ater wh|ch they s|gned an lnormed Consent Form. The study
|n reerence was approved by the Eth|cs Comm|ttee o the Pau||sta
State n|vers|ty (nesp).
II. METHOD
The enro||ed sub|ects were subm|tted to per|phera| b|ood co||ec-
t|on and answered to a deta||ed quest|onna|re about sports and
ACTA OPTOP BPAS 15(1) - 2007
11
(5'-GAACTGC-
CACTTCAGCT-
GTCT, 5'-AGCT-
G C A T T T G -
GAAGTGCTC)
were used, w|th
the |atter be|ng
used as an am-
p|||cat|on |nter-
na| contro|. The
react|on m|xture
was ach|eved w|th 1.5 mM magnes|um ch|or|de, 60 mM Tr|s HC|,
pH 9.0, 15 mM NH
4
SO
4
, dNTPs (200[um]M each), 0,250L Taq po|y-
merase and genom|c DNA at 200ng/500L strength. The matter was
processed |n an automat|c thermocyc|er, be|ng |n|t|a||y subm|tted to
a temperature o 94C or 4 m|nutes or DNA pre-denaturat|on and
then or 40 cyc|es w|th denaturat|on sett|ngs at 94C or 2 m|nutes,
o||me|s r|ng|ng at 59C or 1 m|nute and cha|n extens|on at 72C or
1 m|nute. The |na| extens|on occurred at 72C or 10 m|nutes.
PCP byproducts were subm|tted to e|ectrophores|s |n 2% agarose
ge|, sta|ned w|th eth|d|um brom|de (10mg/mL). The react|on byprod-
ucts were seen as ragments o 480 pb or gene GS77' presence,
215 pb or gene GS7V' presence, and 312 pb or gene CY|'/'. ln
the absence o GS77' and GS7V', mean|ng the nu|| genotype (0/0),
on|y the |ntermed|ate band correspond|ng to gene CY|'/' was seen
(mandatory presence |n a|| |nd|v|dua|s, s|nce a mutat|on o that gene
wou|d be de|eter|ous).

II. 3. StatisticaI anaIysis
The |og|st|c regress|on ana|ys|s was emp|oyed or exam|n|ng the
corre|at|on between smok|ng character|st|cs, BMl, am|||a| h|story
and po|ymorph|sms o the genes vDP, GS77' and V', be|ng a|so
compared by var|ance ana|ys|s. The corre|at|on o the genotypes
w|th the d|erent d|sc degenerat|on degrees (1, 2 or 3) was as-
sessed by Mann-Wh|tney's test. The d|erences between stud|ed
po|ymorph|sms requency and the |n|ury |eve|s o the d|sease (1 or
2 |eve|s) were ca|cu|ated by F|sher's exact test. The t-test was em-
p|oyed or corre|at|ng between mean age at the onset o the d|sease
w|th genotypes and other r|sk actors. For a compar|son between
d|sease |eve|s and sever|ty degrees, genotypes and ||esty|es, the
F|sher's or the Ch|-Squared tests were used. The end resu|ts were
compr|sed |n a con|dence |nterva| o 95% and the P va|ue be|ow
0.05 was cons|dered as stat|st|ca||y s|gn||cant.
RESULTS
I. AIIeIic and Genotypic Frequencies for vDR, GSTT1 and
GSTM1
Tab|e 1 shows the d|str|but|on o a||e||c and genotyp|c requenc|es
or vDP gene |n pat|ents w|th d|scogen|c d|sease and the|r corre-
spond|ng contro|s. The a||e|e requency was s|gn||cant|y h|gher |n
pat|ents (0.46,P~0.0001). The FF genotype was h|gher |n contro|
F/gure 2 - N0c|ea| magne||c |esonance |mage, a| as|a|
p|ane, s|ow|ng d|sc |e|n|a (w|||e a||ow}.
Intervertebral
disc
sub|ects (69.3%) as compared to pat|ents (13.6%, P~0.0001), wh||e
the F genotype was shown to be s|gn||cant|y more requent |n pa-
t|ents (81.8%, P~0.0001). No d|erences were ound |n genotyp|c
requenc|es or GS77' and GS7V' between pat|ents and contro|
sub|ects regard|ng the presence (/) and absence (0/0) o the
gene (P~0.84 and P~0.14, respect|ve|y).
II. PersonaI history
Lumbar pa|n or d|sc hern|a was ment|oned as am|||a| h|story, pre-
erab|y by pat|ents as compared to contro| group (78.8% , 34.1%,
respect|ve|y, P 0.001), accord|ng to Tab|e 2, wh|ch a|so shows
the other character|st|cs, such as smok|ng hab|t and overwe|ght,
s|m||ar between groups.
III. CorreIation between PersonaI History and vDR, GSTT1
and GSTM1 PoIymorphisms
Tab|e 3 shows the corre|at|on between vDP-Fc|| po|ymorph|sm w|th
tobacco use, overwe|ght and am|||a| h|story |n pat|ents and contro|
sub|ects. Frequency, wh|ch has been assessed |n con|unct|on o
at |east one a||e|e (_/) w|th pos|t|ve am|||a| h|story, has shown to
be s|gn||cant|y |ncreased among pat|ents (90%) when compared
to contro| sub|ects (0%, P0.001). On the other hand, the h|gher
requency o FF genotype preva||ed |n smoker pat|ents (80%) when
compared to contro| sub|ects (40%, P0.001).
Pegard|ng the age at d|sease onset, we ound a s|gn||cant|y h|gher
average age |n pat|ents w|th the FF genotype (38 years, 11.9)
when compared to pat|ents w|th genotype _/ (26 years, 10.2, P~
0.013). When th|s character|st|c was assoc|ated to am|||a| h|story,
the mean age at d|sease onset or |nd|v|dua|s w|th the FF a||e|e was
33 years ( 10.7), thus h|gher than those w|th the -/ a||e|e, who
presented a mean age o 26 years ( 10.5). S|m||ar|y, the mean age
at d|sease onset |n |nd|v|dua|s w|th no am|||a| h|story carry|ng the
FF a||e|e was 41 years, 13.3, h|gher than -/ |nd|v|dua|s (28 years,
7.8) (Tab|e 4). No s|gn||cant d|erences were ound between
genotypes and persona| h|story |n pat|ents and contro| sub|ects.
IV. CIinicaI Data and Genotyping for VDR-Fokl, GSTT1 and
GSTM1
Tab|e 5 shows the corre|at|on o vDP-Fc|| genotypes o pa-
t|ents w|th the sever|ty degree and |n|ury |eve|, as assessed
by magnet|c resonance. Genotypes w|th at |east one a||e|e
(_/) showed a corre|at|on w|th the |ncreas|ng sever|ty degree or
d|sc degenerat|on (28.8%) when compared to FF genotype (0.0%,
P~0.0012). The same d|d not happen to |n|ury |eve|s.
There was no corre|at|on between absence o at |east one o the
GS77' and GS7V' genes and |ncreased sever|ty or sp|na| |n|ury
|eve|s.
DISCUSSION
The resu|ts reported |n th|s study express the ex|stence o genet|c
actors on the suscept|b|||ty to |ntervertebra| d|sc degenerat|on. The
AIIeIe Patient ControI P*
N frequency N frequency
F 72 0.54 149 0.85
F 60 0.46 27 0.15 0.0001
Total 1.00 1.00
Genotype N % N %
FF 09 13.6 61 69.3 0.0001
Ff 54 81.8 27 30.7 0.0001
Ff 03 4.6 0 0 0.0767
Total 66 100 88 100
*Fisher`s exact test; N= number of subjects.
Tab/e 1 -D|s|||o0||on o/ a||e||c and geno|jp|c /|eq0enc|es /o| vDP-Fo|/
po|jmo|p||sm |n pa||en|s w||| d|sc degene|a||on and |n con||o| s0ojec|s.
PersonaI
History
Patient ControI
P*
N
Frequency
%
N
Frequency
%
Smoking
habit
20 30.3 15 17.0 0.055
Overweight** 10 15.5 21 23.9 0.224
Familial
history
52 78.8 30 34.1 <0.001
*Test = logistic regression; ** Overweight: BMl=25 - 30kg/m.
Tab/e 2 - D|s|||o0||on o/ pe|sona| ||s|o|j o/ pa||en|s w||| d|sc degene|a||on
and o/ con||o| s0ojec|s.
ACTA OPTOP BPAS 15(1) - 2007
12
a||e||c and genotyp|c d|str|but|on or Fc|| po|ymorph|sm o the v|tam|n-D
receptor gene d|erent|ates pat|ents rom contro| sub|ects, s|nce the
s|gn||cant|y |ncreased requency o a||e|e |n pat|ents w|th d|sc degen-
erat|on (0.46) was outstand|ng as compared to contro| sub|ects (0.15).
The F genotype was present |n most o the sub|ects aected by the
d|sease (81.8%), wh||e |n contro| group there was a preva|ence o the
dom|nant FF homozygote (69.3%).
The requency o genotypes or vDP-Fc|| |n these popu|at|ons var|es
between the d|erent ethn|ca| groups, w|th FF requenc|es rang|ng rom
28% to 35%, F rom 48% to 50% and rom 14 to 17% among wh|te
or Caucas|an |nd|v|dua|s aged between 20 and 62 years
(3,14,16)
. On the
other hand, among b|ack or Ar|can popu|at|on, the FF genotype |s
preva|ent (66%), o||owed by F (31%) and (4%)
(14)
. The numbers or
contro| group |n th|s study were 69.3%, 30.7% and 0.0%, respect|ve|y,
w|th the |ow requency ound or a||e|e potent|a||y be|ng exp|a|ned as a
resu|t o a sma|| case ser|es, added by the |arge and rea| m|scegenat|on
o the Braz|||an popu|at|on.
v|deman et a|.
(3)
po|nt to a stra|ght corre|at|on between an |ncreased
lDD degree and the presence o each a||e|e . On th|s ground, |nd|v|du-
a|s carry|ng the genotype showed to be most aected, decreas|ng|y
o||owed by F and FF genotypes. Cons|stent|y, |n th|s study, the a||e|e
has shown to be corre|ated to a h|gher degree o degenerat|on when
compared to a||e|e F (P~0.0012).
The onset o degenerat|ve process |n young pat|ents strong|y suggests
the |n|uence o genet|c components
(4)
. Such corre|at|on resu|ts rom the
act that, |n those |nd|v|dua|s, the |ntervertebra| d|sc has been exposed to
env|ronmenta| r|sk actors or a shorter per|od when compared to o|der
|nd|v|dua|s
(5)
. Our data corroborate th|s corre|at|on, s|nce the age at
d|sease onset was |ower among pat|ents carry|ng the F or genotype
(average: 26 10.2 years), cons|der|ng the vDP-Fc|| po|ymorph|sm,
wh||e the average or pat|ents w|th the FF genotype was 38 11.9
years. lnterest|ng|y, bes|des the ear|y |nvo|vement o pat|ents carry|ng
the a||e|e , d|scogen|c d|sease h|story |n pat|ents' am|||es presented
a s|m||ar pro||e. When th|s character|st|c was corre|ated to pat|ents'
am|||a| h|story and a|so corre|ated to the presence o the a||e|e , the
mean age at d|sease onset was |ower (26 10.5 years) |n pat|ents w|th
pos|t|ve am|||a| h|story, wh||e the average or |nd|v|dua|s w|th no am|||a|
h|story o d|sc degenerat|on was 33 11.7 years (P~ 0.025).
Thereore, the presence o d|scogen|c d|sease |n the am||y was ound
|n the ma|or|ty o the pat|ents |n the study group (78.8%), as oppos|te
to contro| sub|ects, where th|s requency has shown to be reduced
(34.9%). From the tota| number o pat|ents w|th pos|t|ve am|||a| h|story,
27% had the a||e|e , wh||e none o the |nd|v|dua|s |n contro| group pre-
sented w|th th|s a||e|e, even w|th pos|t|ve am|||a| h|story or the d|sease.
Thus, our |nd|ngs re|norce the strong |mportance o genet|cs on the
suscept|b|||ty to d|sc degenerat|on.
The contr|but|on g|ven by vDP Fc|| po|ymorph|sm or the d|sease
may be exp|a|ned by a structura| mod||cat|on o the vDP prote|n
due to a change on the s|te start|ng exon 2 transcr|pt|on. The F a|-
|e|e does not have the |rst ATG start|ng s|te, thereore, transcr|pt|on
starts at the second s|te, produc|ng a shorter prote|n |n three am|no
ac|ds. Oppos|te|y, th|s does not happen w|th the a||e|e , thereore,
the resu|t|ng prote|n has a norma| s|ze. Jurutka et a|.
(7)
showed that
the sma||er a||e|e, w|th 424 am|no ac|ds (a||e|e F) |nteracts more
e|c|ent|y w|th the transcr|pt|on actor TFllB, |ead|ng them to the
conc|us|on that th|s a||e|e presents a h|gher transcr|pt|ona| power,
a act that was a|so corroborated by Chen et a|.
(17)
and |ter||ndem
et a|.
(6)
. The act|v|ty d|erence rat|o or both prote|ns seems to be
corre|ated to the change on |ts ab|||ty to bond to the act|ve orm
o v|tam|n D or, subsequent|y, attach|ng to ret|no|c ac|d receptor
(PXP) and to the v|tam|n-D respons|ve e|ement (vDPE) and, |na||y,
act|vate transcr|pt|on
(8)
. Thus, the add|t|on o three am|no ac|ds to
v|tam|n-D receptor's prote|n |s ab|e to mod|y the who|e v|tam|n-D
act|on comp|ex, resu|t|ng |n transcr|pt|ona| process a||ure.
ln add|t|on to v|tam|n-D |nvo|vement on d|erent|at|on, pro||erat|on,
maturat|on o cart||ag|nous ce||s and |ts |n|uence on proteog|ycans
synthes|s
(5)
, v|tam|n D3 was proven to part|c|pate on ce|| pro||erat|on
and growth by st|mu|at|ng the |nsu||n-dependent growth actor l (lGF-
l) and |ts receptor
(18)
. Adm|tted|y, v|tam|n D3 act|vates target-genes
promoter through mu|t|p|e-mo|ecu|es comp|ex. lt |s suggested that
v|tam|n D3 act|vates the lGF-l gene promoter |ocated near to the gene
or v|tam|n D receptor. lGF-l |s expressed on |ntervertebra| d|sc t|ssue
and st|mu|ates proteog|ycans synthes|s on pu|pous nuc|eus ce||s
(5)
.
Thus, changes on v|tam|n-D receptors express|on wou|d |mpa|r |nter-
vertebra| d|sc's homeostas|s.
A|though the genet|c actor has been po|nted out as a ma|or |n|uenc|ng
actor on d|sc degenerat|on process, other actors seem to |n|uence
changes on norma| metabo||sm o the d|sc
(4)
. From these, we cou|d
ment|on env|ronmenta| actors, represented by phys|ca| and occupa-
t|ona| act|v|t|es, as we|| as ||esty|es (such as d|et and tobacco use),
among others, a|though v|deman e Batt|e
(19)
had conc|uded that the
env|ronment cou|d exp|a|n on|y a sma|| port|on o th|s d|sease. Our
|nd|ngs seem to be cons|stent w|th th|s |dea, once no corre|at|on was
ound between pat|ents' occupat|ona| act|v|t|es and pos|t|ve s|gns o
degenerat|on.
Another env|ronmenta| actor has been corre|ated to sp|na| cond|t|ons
|n many ep|dem|o|og|ca| stud|es
(20)
. Obes|ty |s common among pa-
t|ents subm|tted to orthopaed|c surg|ca| procedures, among others,
the remova| o a hern|ated d|sc. However, Leboeu
(21)
, when conduct|ng
a rev|ew on th|s matter ound that on|y 21 among 65 stud|es showed
a pos|t|ve corre|at|on between overwe|ght/ obes|ty and sp|na| cond|-
t|ons. S|m||ar|y, our |nd|ngs showed no such corre|at|on, even when
the presence o a||e|e was compared to overwe|ght |n pat|ents and
contro| sub|ects (P~0.688).
Smok|ng hab|t |s a urther r|sk actor to be added to the ||st o e|ements
suspected to have de|eter|ous eects on |ntervertebra| d|sc. Many
PersonaI
History
Patient ControI
F/F _/f F/F _/f
N % N % N % N % P*
Smoking
habit
16 80.0 4 20.0 6 40.0 9 60.0 <0.001
Overweight**
1 10.0 9 90.0 18 85.7 3 14.3 0.688
Familial
history
3 10.0 27 90.0 30 100.0 0 0.0 <0.001
*Test = logistic regression; ** Overweight: BMl=25 - 30kg/m.
7ao|e 3 Gene||c po|jmo|p||sms /o| vDP-Fo|/ and pe|sona| ||s|o|j |n pa||en|s
w||| d|sc degene|a||on and |n con||o| s0ojec|s.
VDR
Genotype
Onset of Disease (age-years)
GeneraI SD*** Presence
of FH**
SD*** Absence
of FH**
SD***
FF 38 11,9 33 11,7 41 13,3
/f 26 10,2 26 10,5 28 7,8
P* 0,013 0,025
P*=t-test;** Familial History (FH);*** Standard Deviation (SD)
7ao|e 4 Co||e|a||on oe|ween age a| ||e onse| o/ d|sease and /am|||a| ||s|o|j
|n pa||en|s w||| d|sc degene|a||on.
Genotype
Severity* Injury LeveI**
1
N %
2
N %
3
N %
1
N %
2
N %
F/F 4
7.7
26 50.0 0 0.0 4 7.7 2 3.9
_/f 4
7.7
3 5.8 15 28.8 15 28.8 31 59.6
P*=0.0012 (Mann-Whitney`s test); P**=0.175 (Fisher`s test)
7ao|e 5 Co||e|a||on oe|ween vDP-Fo|/ Po|jmo|p||sm, sete|||j o/ d|sc
degene|a||on, and |nj0|j |ete|.
ACTA OPTOP BPAS 15(1) - 2007
13
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|nvest|gat|ons have documented the |ncreas|ng |nc|dence o |umbar
and sc|at|c pa|n, as we|| as d|sc degenerat|on |n chron|c smokers
compared to non-smokers
(22)
. Exper|ments conducted |n |so|ated
ce||s o the pu|pous nuc|eus reported that c|garette components, |n
add|t|on to reduce d|sc vascu|ar|zat|on, promote both a decreased
g|ycosam|nog|ycans product|on and the express|on o type-ll co||agen
gene. Other aspects assoc|ated to degenerat|on have been reported,
such as reduced number o pu|pous nuc|eus ce||s
(9)
. As a resu|t o
the changes on g|ycosam|nog|ycans content, the pu|pous nuc|eus
dehydrates, chang|ng the norma| d|sc hydrostat|cs and, consequent|y,
|ts damp|ng propert|es.
Our resu|ts showed that s|gn||cant|y |ncreased requenc|es o FF and
reduced requenc|es o -/ were preerab|y observed |n smoker pat|ents
when compared to contro| sub|ects. ln th|s case, |t |s not|ced that these
pat|ents, even w|th the most act|ve genotype |n v|tam|n D capture,
became more vu|nerab|e when exposed to tobacco.
ln th|s study, the assessment o the |n|uence o po|ymorph|sms |n
genes |nvo|ved w|th c|garette components' metabo||sm - GS77' and
GS7V' on d|sc degenerat|on has shown that 21.2% o pat|ents and
19.3% o contro| sub|ects d|d not carry T1 (GS77' 0/0), wh||e or M1,
nu|||ty (GS7V' 0/0) among pat|ents was 47%, and 34.1% |n contro|
sub|ects, w|th no d|erence between stud|ed groups (P~ 0.84 and 0.14
respect|ve|y). Nu|||ty requenc|es or genes GS7V' (34.1%) and GS77'
(19.3%) |n contro| sub|ects are cons|stent to Poss|t et a|.
(23)
.
Our resu|ts show that the a||e|e can be cons|dered as a r|sk ac-
tor or the ear|y onset o lDD s|gns. Furthermore, the smok|ng hab|t
seemed to |nterere on the process, regard|ess o the presence o a
avorab|e genotype or genes |nvo|ved |n the detox||cat|on o smoke
components.
CONCLUSIONS
1. The a||e|e or v|tam|n D receptor (vDP-Fc|') |s assoc|ated to
|ntervertebra| d|sc degenerat|on, preerab|y |n pat|ents w|th am|||a|
h|story o the d|sease, however, th|s does not occur w|th genet|c
po|ymorph|sms or g|utath|one-S-transerase enzymes (GS77'
and GS7V').
2. The smok|ng hab|t has shown to be a r|sk actor, even or pat|ents
w|th the FF genotype o vDP-Fc|', po|ymorph|sm, regard|ess o
the genotypes or detox||cat|on genes (GS7V' or 7'), wh|ch |n
con|unct|on w|th |nd|v|dua| character|st|cs, |nc|ud|ng overwe|ght,
am|||a| h|story and sever|ty o the d|sease do not show corre|at|on.
On the other hand, d|sease sever|ty and |ts ear|y onset are cor-
re|ated to genotypes _/.
3. The am|||a| h|story or d|sc degenerat|on |s reported to be a strong
r|sk actor among pat|ents, suggest|ng genet|c suscept|b|||ty to the
d|sease, w|th the same not occurr|ng concern|ng anthropometr|c,
env|ronmenta| and occupat|ona| actors.
ACKNOWLEDGEMENT
Th|s study was |nanc|a||y supported by CNPq (Nat|ona| Counc|| or
Sc|ent||c and Techno|og|ca| Deve|opment) and by FNDNESP
(NESP Pesearch Support Foundat|on).