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A nonconservative, coding single-nucleotide polymorphism in the N-

terminal region of lactoferrin is associated with aggressive periodontitis in an
African-American, but not a Cauca...

Article  in  Genes & Immunity · November 2005

DOI: 10.1038/sj.gene.6364239 · Source: PubMed

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 Genes and Immunity (2005) 6, 632–635
& 2005 Nature Publishing Group All rights reserved 1466-4879/05 $30.00
www.nature.com/gene

BRIEF COMMUNICATION
A nonconservative, coding single-nucleotide
polymorphism in the N-terminal region of lactoferrin
is associated with aggressive periodontitis in an
African-American, but not a Caucasian population
WJ Jordan, J Eskdale, GP Lennon, R Pestoff, L Wu, DH Fine and G Gallagher
Department of Oral Biology, University of Medicine and Dentistry of New Jersey, Newark, NJ, USA

Lactoferrin is an antimicrobial protein which plays an important role in regulating bacteria that are associated with aggressive
periodontitis. Lactoferrin kills directly (via its strongly cationic N-terminal region) and indirectly, through sequestering the iron
that bacteria require for growth. As aggressive periodontitis has a strong heritable component, we hypothesized that genetic
variation within the lactoferrin gene may play a role in susceptibility to this condition. We have identified and examined a novel,
functional, single-point A/G nucleotide mutation causing a threonine/alanine substitution at position 11 (T11A) of the secreted
lactoferrin protein. In a pilot case-controlled study of aggressive periodontitis, analysis of 46 African-American patients and 78
controls showed that patients were twice as likely to express the G nucleotide (alanine) allele over controls (60.3 vs 30.4%;
P ¼ 0.0007, odds ratio ¼ 2.564, 95% CI ¼ 1.475–4.459). A Caucasian population of 77 patients and 131 controls showed no
such association (P ¼ 0.5201, odds ratio ¼ 0.862, 95% CI ¼ 0.548–1.356). The data presented provide a new insight into the
genetic susceptibility to aggressive periodontitis.
Genes and Immunity (2005) 6, 632–635. doi:10.1038/sj.gene.6364239; published online 6 October 2005

Keywords: lactoferrin; polymorphism; aggressive periodontitis; African American

Lactoferrin is an 80 kDa cationic protein with strong
antibacterial and immunomodulatory functions.1–5 The
antibacterial properties were originally attributed solely
to its ability to bind the iron necessary for bacterial
growth. However, it has been established more recently
that lactoferrin also binds to bacteria and kills through
direct interactions governed by its strongly basic N-
terminal region.6–9 In addition to its ability to kill
bacteria, lactoferrin can also neutralize endotoxin and
inhibit the induction of NFkB in monocytes in response
to LPS, resulting in lowered IL-6 and TNF-alpha
production.10–12
Lactoferrin is present in high concentrations in saliva
and is thought to play a particularly important role in
regulation of those bacteria present within the oral cavity.
This protein regulates several bacteria that are associated
with periodontal disease, including Porphyromonas gingi-
valis (Pg), Prevotella intermedia (Pi), Prevotella melanino-
genica (Pm) and Actinobacillus actinomycetemcomitans
(Aa).13–18 Lactoferrin’s effects on Aa, which is considered
to have the strongest association with aggressive period-
Correspondence: Dr G Gallagher, Department of Oral Biology, New Jersey
Dental School, 185 South Orange Avenue, MSB, Room C636, Newark, NJ
07103-2714, USA. E-mail: gallaggr@umdnj.edu
Received 15 December 2004; revised 9 May 2005; accepted 9 May
2005; published online 6 October 2005
ontitis, occurs through direct killing, as well as through
inhibition of attachment to epithelial cells and the
development of biofilms.14,19,20
Studies examining the pattern of inheritance of
aggressive periodontitis in families suggest a genetic
basis for susceptibility.18,21 The disease is particularly
common within African-American populations, being up
to 15 times more prevalent than in Caucasians.21 As
lactoferrin has been shown to be active against a number
of pathogenic bacteria, including Aa, we hypothesized
that single-nucleotide polymorphism (SNPs) within
the lactoferrin gene could play a role in the genetic
susceptibility that is seen to this organism and aggressive
periodontitis. One recent study has identified two
variants of lactoferrin and demonstrated that these
variants exhibit different antibacterial and transcrip-
tional activation activities.22 Examination of a small
population of periodontal patients suggested that these
differences may go some way in explaining susceptibility
to periodontal disease. However, from studying different
ethnic populations, the data could not help explain
why African Americans appear to be more susceptible
to periodontitis due to this organism.22 In the present
report, we examine further the genetic associations
between this gene and aggressive periodontal disease
in both African-American and Caucasian populations,
using a novel, nonconservative, SNP in the human
lactoferrin gene.

We examined the SNP database for the presence of
polymorphisms within the region of the human lacto-
ferrin gene which encodes the functionally important,
strongly basic, N-terminal region.5 By altering the amino-
acid composition, such SNPs may potentially affect the
antimicrobial properties of lactoferrin and thus be
important in disease caused by bacteria, including
periodontitis. The search revealed a novel A/G poly-
morphism at nucleotide 88 of the lactoferrin gene,
resulting in a threonine/alanine substitution at position
11 (T11A) of the mature peptide. This substitution was of
interest not only for its location within the strongly basic
N-terminal region of the lactoferrin molecule that
mediates its antibacterial properties, but also due to the
nature of the amino acids involved in the substitution
itself. Threonine is polar and commonly found in protein
functional centers, being able to form hydrogen bonds
with a variety of polar subsrates, while alanine is a
nonpolar, nonreactive amino acid that is rarely involved
in protein function. Threonine is also commonly found
to play a role in phosphorylation of proteins (as with
serines and tyrosines) in order to facilitate signal
transduction processes. This polymorphism therefore
represented a strong candidate as a regulator of
lactoferrin function, through the substitution of threo-
nine by alanine.
We used PCR-restriction fragment length polymorph-
ism (PCR-RFLP) analysis of this Lactoferrin þ 88 A/G
polymorphism in African-American and Caucasian
aggressive periodontitis patients in comparison to
ethnically matched controls (Table 1). PCR amplification
of a 720 bp region spanning the polymorphic site was
performed on patient and control DNA. The products
were digested overnight at 371C with 2.5 U of ApaL I
restriction endonuclease and fragments separated using
agarose gel electrophoresis. Homozygous G allele ap-
pears as 720 bp, while the homozygous A allele appears
as bands of 468 and 252 bp. Heterozygotes displayed all
three bands of 720, 468 and 252 bp. Diagnosis of
aggressive periodontitis was made on clinical, radio-
graphic and historical findings, which show rapid
attachment loss and bone destruction as previously
described.23 Full medical histories were recorded to rule
A nonconservative, coding single-nucleotide polymorphism
WJ Jordan et al
633
out conditions which might predispose to periodontal
disease. The control ‘healthy’ populations were unre-
lated individuals, ethnically and geographically matched
to the study groups having no known autoimmune or
malignant disease, nor were they relatives of such
patients.
The data revealed a significant over-representation of
the G (alanine) allele in the African-American period-
ontitis patients (Table 1; P ¼ 0.0007, odds ratio ¼ 2.564,
95% CI ¼ 1.475–4.459). No association was found in the
Caucasian population (P ¼ 0.5201, odds ratio ¼ 0.862,
95% CI ¼ 0.548–1.356), suggesting that this polymor-
phism may be an important factor in the previously
observed susceptibility differences between different
ethnic populations.18,21
Further analysis showed that the genotype was also
significantly associated with disease in the African-
American population (Table 2; P ¼ 0.0038); both ‘G’-
containing genotypes were elevated in patients over
controls. The frequency of the A/A genotype was twice
as likely to be found in controls over patients (60.3 vs
30.4%), while the A/G genotype was 1.6 times more
likely to be found in patients over controls (52.2 vs 33.3%)
and the G/G genotype 2.7 times more common in
patients over controls (17.4 vs 6.4%). Again, no cor-
relation was evident between genotype and disease in
the Caucasian population (Table 2; P ¼ 0.4208). Analysis
of grouped ‘G’-containing genotypes against the A/A
genotype using w2 analysis showed a strong correlation
with disease (P ¼ 0.0013, odds ratio ¼ 3.465, 95% CI ¼
1.597–7.520), while the grouped ‘A’-containing genotypes
analyzed against the G/G genotype were not signifi-
cantly associated with disease (P ¼ 0.0538, odds ratio ¼
3.074, 95% CI ¼ 0.941–10.045). Therefore, it appears that
it is the presence of the G allele (or absence of A) that
is associated with disease rather than the presence of A
(or absence of G) being protective.
Analysis of the two normal control populations
showed differences in allele distribution of the lactoferrin
þ 88 A/G polymorphism between the African American
and the Caucasians. Although the G allele was associated
with aggressive periodontitis in African-American
patients, Caucasian normal controls had a very high

Table 1 Distribution of Lactoferrin nucleotide +88 nucleotide allele frequencies in African-American and European Caucasian aggressive
periodontitis patients and ethnically matched controls

Allele African Americans Caucasian

Controls Patients Controls Patients

A 120/156 (76.9%) 52/92 (56.5%) 64/262 (24.4%) 42/154 (24.4%)

G 36/156 (23.1%) 40/92 (43.5%) 198/262 (75.6%) 112/154 (72.7%)

P ¼ 0.0007 P ¼ 0.5201

The Lactoferrin +88 A/G polymorphism was discriminated by PCR and restriction enzyme digestion. Primer pairs for detection of the
Lactoferrin +88 A/G mutation were 50 -gaaccacagacctctagccaat-30 and 50 -cttttggaggatttccctctct-30 . PCR products were digested overnight at
371C with 2.5 units of ApaL I restriction endonuclease and digested fragments separated and visualized in 3% (w/v) agarose gel. The
homozygous G allele appears as a visible band of 720 bp, while the homozygous A allele appears as bands of 468 and 252 bp. Heterozygotes
appeared as all three bands 720, 468 and 252 bp. DNA from 46 African-American patients and 78 ethnically and geographically matched
healthy subjects were examined as controls along with 77 Caucasian generalized AP (GAP) patients and 131 controls. Allele and genotype
frequencies were assessed statistically using Pearson’s w2 test.

Genes and Immunity

 A nonconservative, coding single-nucleotide polymorphism
WJ Jordan et al
634
Table 2 Frequencies of Lactoferrin (+88 A/G) SNP genotypes in African-American and European Caucasian aggressive periodontitis
patients and matched controls
Genotype African Americans
Controls
A/A 47/78 (60.3%)
A/G 26/78 (33.3%)
G/G 5/78 (6.4%)
P ¼ 0.0038
frequency of this allele, yet did not have the disease.
Interestingly, animal models of SLE have revealed that
both susceptibility and resistance loci exist, which
combine to give the overall likelihood of disease.24 Such
a mechanism may explain the difference in disease
prevalence between African-American individuals and
Caucasians, if Caucasians were to have one or more
active resistance loci. Alternatively, hitherto undefined
loci which control the presence of Aa may render African
Americans more susceptible to infection than Cauca-
sians, leading to greater disease development. Thus, it
would seem that this particular polymorphism alone
does not fully explain susceptibility, and it is likely that
further polymorphisms, possibly within the lactoferrin
gene, may play a role in susceptibility to aggressive
periodontal disease. Further studies will be required to
elucidate the mechanism linking the polymorphism and
disease, examining the possible functional role of this
polymorphism in dictating the biological activity of
lactoferrin. One potential confounding factor in the data
presented is the possibility of admixture in the African-
American population studied. In order to address this, a
larger study with ethical permission to permit genetic
analysis of geographical origin would be necessary. The
study of larger groups of patients and controls from
other ethnicities, including those in different geographi-
cal regions, would also be useful in enhancing the
findings presented here.
A number of studies have examined links between
polymorphisms within host-response factors and aggres-
sive periodontitis.25 These include examination of
genes encoding inflammatory cytokines such as IL-1
and TNF-a, the anti-inflammatory cytokine IL-10 and the
Fc-gamma receptor. The data thus far from these studies
appear to be controversial, with a number of studies
revealing associations and others showing contradictory
data. As with most diseases that have complex etiology,
the genetic factors that contribute to periodontal disease
susceptibility and progression appear to be numerous.
The results presented here show a novel strong associa-
tion with the lactoferrin gene, adding to the growing
evidence that genes play a role in the predisposition to
and progression of periodontal disease.18,21,25–27
The data presented in this study describe a novel,
functional single-nucleotide polymorphism that goes
some way to help explain the genetic susceptibility
associated with aggressive periodontitis in African
Americans. It may be that the polymorphism described
Genes and Immunity
Caucasian
Patients Controls Patients
14/46 (30.4%) 6/131 (4.6%) 7/77 (9.1%)
24/46 (52.2%) 52/131 (39.7%) 28/77 (36.4%)
8/46 (17.4%) 73/131 (55.7%) 42/77 (54.5%)
P ¼ 0.4208
here affects the ability of lactoferrin to bind to and/or kill
different genetic variants of bacteria, including Aa, that
have previously been implicated with the pathogenicity
in aggressive periodontitis.4,16,17,28–31
Acknowledgements
This study was supported by the National Institutes of
Health, National Institute of Dental and Craniofacial
Research grant number 1R21DE014997, by the Health
Disparities Program (WJJ and JE) and by the New Jersey
Dental School (GPL and JE).
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