Genes and Immunity (2005) 6, 632–635 & 2005 Nature Publishing Group All rights reserved 1466-4879/05 $30.00 www.nature.com/gene
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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 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 Nterminal 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 gingivalis (Pg), Prevotella intermedia (Pi), Prevotella melaninogenica (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:
[email protected] 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 transcriptional 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.
A nonconservative, coding single-nucleotide polymorphism WJ Jordan et al
We examined the SNP database for the presence of polymorphisms within the region of the human lactoferrin gene which encodes the functionally important, strongly basic, N-terminal region.5 By altering the aminoacid 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 polymorphism 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 threonine by alanine. We used PCR-restriction fragment length polymorphism (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 appears 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, radiographic and historical findings, which show rapid attachment loss and bone destruction as previously described.23 Full medical histories were recorded to rule
out conditions which might predispose to periodontal disease. The control ‘healthy’ populations were unrelated 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 periodontitis 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 polymorphism 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 AfricanAmerican 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 correlation 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 significantly 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
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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
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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
Caucasian
Controls
Patients
Controls
Patients
A/A
47/78 (60.3%)
14/46 (30.4%)
6/131 (4.6%)
7/77 (9.1%)
A/G
26/78 (33.3%)
24/46 (52.2%)
52/131 (39.7%)
28/77 (36.4%)
G/G
5/78 (6.4%)
8/46 (17.4%)
73/131 (55.7%)
42/77 (54.5%)
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 Caucasians, 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 AfricanAmerican 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 geographical 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 aggressive 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 association 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
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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