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Ethnic Differences of Polymorphisms in Cytokine and Innate Immune System Genes in Pregnant Women

Daniel P. Nguyen, MD^*, Mehmet Genc, MD, PhD^*, Santosh Vardhana^*, Oksana Babula, MD^*, Andrew Onderdonk, PhD and Steven S. Witkin, PhD^*

From the *Division of Immunology and Infectious Diseases, Department of Obstetrics and Gynecology, Weill Medical College of Cornell University, New York, New York; and Department of Obstetrics, Gynecology and Reproductive Biology and Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts.

Address reprint requests to: Dr. Steven S. Witkin, Department of Obstetrics and Gynecology, Weill Medical College of Cornell University, 525 East 68th Street, Box 35, New York, NY 10021; e-mail: switkin{at}med.cornell.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
OBJECTIVE: Investigations of the possible role of polymorphic genes in pregnancy outcome may be influenced by ethnic variations in genotype or allele frequencies. Differences in allelic carriage of immune system-related genes among white, black, and Hispanic pregnant women living in New York City and Boston were evaluated.

METHODS: DNA was extracted from buccal or vaginal epithelial cells collected from 198 white, 75 black, and 114 Hispanic pregnant women who delivered at term and who had no history of a preterm birth. Genetic polymorphisms in the immunoregulatory genes encoding interleukin (IL)-1ß, tumor necrosis factor-, IL-4, IL-10, IL-1 receptor antagonist (IL-1ra), mannose-binding lectin, toll-like receptor-4, and the 70-kDa heat shock protein were determined.

RESULTS: Allele 2 of the IL-1ra gene (IL1RN*2) and IL-4 –590C homozygosity were 4-fold less common in blacks than in whites or Hispanics (P < .001). The IL-4 –590T allele was almost 2-fold more common in Hispanics than in whites (P < .001). The frequency of the 70-kDa heat shock protein 1267G allele was at least 1.4 times greater in blacks compared with whites (P < .001) or Hispanics (P = .002), whereas the homozygous mannose-binding lectin codon 54G allele was observed at least 4.5 times more often in Hispanics compared with whites (P = .007) or blacks (P = .02).

CONCLUSION: Investigations of the role of genetic factors affecting pregnancy outcome must be cognizant of ethnic variations when enrolling case and control subjects for studies on allele and genotype frequencies.

LEVEL OF EVIDENCE: III

The genes encoding pro- and anti-inflammatory cytokines that regulate the magnitude of proinflammatory immune activity, as well as components of the antimicrobial innate immune defense system, are polymorphic. Possession of distinct alleles of polymorphic genes could account for individual and ethnic differences in the rate and extent of production of individual proteins as well as variations in the acquisition and/or severity of a particular disorder. Possession of the tumor necrosis factor- (TNF-) –308 G>A polymorphism in the promoter region of the gene has been associated with preterm premature rupture of the fetal membranes (PROM) in blacks³; allele 2 of the 86-base pair repeat polymorphism in intron 2 of the IL-1 receptor antagonist (IL-1ra) gene has been associated with preterm PROM in Hispanics⁴ and in multifetal pregnancies;⁵ an IL-1ß +3953 C>G polymorphism in exon 5 has been associated with preterm birth in blacks,⁴ and the IL-4 –590 C>T promoter polymorphism has been related to an increased rate of spontaneous preterm birth in multifetal pregnancies.⁶ Allelic distinctions in cytokine genes also have been shown to differentially influence allograft survival after organ transplants,^7,8 a situation that may be somewhat analogous to the survival of a semiallogeneic fetus in a pregnant woman after immune system perturbation.

Components of the innate immune system comprise the initial defense against invading microorganisms. Because cell-mediated immune defense is down-regulated in pregnant women,⁹ polymorphisms in genes regulating the extent of innate immune activation may increasingly influence susceptibility to microbial invasion during pregnancy. Mannose-binding lectin plays an important role in innate immunity. This circulating protein binds to mannose and N-acetylglucosamine residues on microbial surfaces, leading to complement activation and opsonization of the microorganisms by phagocytic cells. The mannose-binding lectin gene is polymorphic in exon 1 and the variant alleles produce an unstable mannose-binding lectin protein. This results in decreased mannose-binding lectin serum levels and an increased susceptibility to infections.¹⁰

Toll-like receptor-4 is a membrane-bound protein that recognizes the lipopolysaccharide (LPS) of gram-negative bacteria. Binding of LPS to toll-like receptor-4 initiates a cascade of events culminating in proinflammatory cytokine gene activation.¹¹ The toll-like receptor-4 +896 A>G polymorphism in exon 4 of the gene has been associated with reduced LPS binding capacity.¹² One study found no relationship between this polymorphism in the fetus and the rate of preterm PROM in blacks,¹³ whereas a second study from Finland did observe a relationship between toll-like receptor-4 +896G and preterm birth.¹⁴

Synthesis of the inducible 70-kDa heat shock protein is greatly up-regulated when cells are confronted with adverse stimuli such as infection or inflammation. Intracellularly, the 70-kDa heat shock protein binds to other proteins and prevents their degradation and incorrect assembly during stressful conditions. The 70-kDa heat shock protein also inhibits apoptosis by binding to p53 and c-myc and blocking activation of the caspase cascade.^15,16 Extracellular 70-kDa heat shock protein induces proinflammatory cytokine production and acts as an early warning signal that a potential danger is present.¹⁷ A +1267 A>G polymorphism in the 70-kDa heat shock protein gene has been associated with reduced messenger RNA production.¹⁸

Studies of associations between polymorphisms in immune system-related genes and pregnancy outcomes may be confounded if allele frequencies differ between ethnic groups in the patient and/or the control population under examination. In this study, we report on variations in the rate of polymorphisms in proinflammatory (TNF-, IL-1ß), anti-inflammatory (IL-1ra, IL-4, IL-10), and innate immune system (toll-like receptor-4, mannose-binding lectin, 70-kDa heat shock protein) genes in white, black, and Hispanic pregnant women in New York City and Boston.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The study group was composed of 387 women with uncomplicated pregnancies and deliveries at more than 37 weeks of gestation from The New York Presbyterian Hospital in New York and Brigham and Women's Hospital in Boston between January 2000 and March 2002. Mothers with a history of preterm labor or incompetent cervix and those who gave birth to babies with anomalies, on the basis of chart review, were excluded. The ethnic breakdown was 198 white, 75 black, and 114 Hispanic women based on self-reporting by the mother. In this investigation, intraracial differences within each group were not evaluated. All second-trimester pregnant women who met the eligibility criteria were selected randomly for entry into the study without regard to ethnicity. The study was approved by the institutional review boards of both medical centers.

Buccal (The New York Presbyterian Hospital) or vaginal (The New York Presbyterian Hospital and Brigham and Women's Hospital) mucosal epithelial cells were used as sources of maternal DNA, the choice being determined by the attending obstetrician. Because the DNA found in all cells from an individual is identical, the source of cells was immaterial. Before collecting buccal samples, we asked the subjects to rinse their mouths with water. Buccal samples were then obtained by rotating a cotton swab (Epicenter; Madison, WI) against the inner side of the cheek. The swabs were shaken into 1.0-mL microcentrifuge tubes containing 0.5 mL of phosphate-buffered saline and stored at –20°C. Vaginal lavage samples were obtained by instilling 10 mL of saline into the vagina, aspirating the fluid from the posterior fornix, and transferring it to a 15-mL tube. The samples were centrifuged at 600g for 14 min and the supernatants stored at –70°C. Samples from Brigham and Women's Hospital were shipped to New York on dry ice for gene polymorphism analysis.

Samples were thawed, microcentrifuged, and the pellets washed 3 times in phosphate-buffered saline. Cell lysis and release of DNA were accomplished by resuspending the cell pellet in a Brij 35 detergent and proteinase K–containing buffer, as described previously.⁴ The proteinase K was subsequently inactivated by incubation of the samples at 95°C for 10 minutes; aliquots were stored at –80°C until tested.

Aliquots (25 µL) of the lysed samples were combined with an equal volume of 10 mmol/L Tris[hydroxymethyl]aminomethane, hydrochloride that contained 1.5 mmol/L MgCl₂, 50 mmol/L potassium chloride, 0.2 mmol/L each of deoxyadenosine 5'-triphosphate, 2'-deoxycytidine 5'-triphosphate, 2'-deoxyguanosine 5'-triphosphate, and thymidine triphosphate, 1.25 U of TaqDNA polymerase, and 30 pmol of primer pairs specific for the polymorphic region of the gene to be analyzed.

Analysis for length variants in intron 2 of the IL-1ra gene was as described.^4,19 The polymerase chain reaction products were identified on a 1% agarose gel after ethidium bromide staining. The 410-base pair product is allele 1 (IL1RN*1), and the 240-base pair product is allele 2 (IL1RN*2).

Analysis for the IL-1ß +3953 C>G polymorphism, which contains a Taq I restriction site, was as described.^4,20 Taq I digestion of the 249-base pair amplicons resulted in either 2 fragments of 135 and 114 base pairs (IL1B +3953C) or an intact 249-base pair fragment (IL1B + 3953G).

Analysis of the TNF –308 G>A promoter polymorphism, which contains an NcoI restriction site, was performed by following a published protocol.²¹ Digestion resulted in formation of either 2 87- and 20-base pair fragments (TNF –308 G) or 1 uncut 107-base pair fragment (TNF –308 A).

The IL-4 –590 C>T promoter polymorphism, which contains a BsmFI restriction site, was determined by using a published protocol.²² Digestion resulted in 2 192- and 60-base pair fragments (IL4 –590 C) or an uncut 252-base pair fragment (IL4 –590 T).

The IL-10 –1082 A>G promoter polymorphism containing a Mn1I restriction site was assessed as described.²³ IL10 –1080 A yielded 2 fragments of 133 and 65 base pairs, whereas IL10 –1082 G yielded 3 fragments of 94, 65, and 39 base pairs.

The region encompassing the TLR4 +896 A>G polymorphism and a NcoI digestion site was amplified and identified as described.¹⁴ TLR4 +896A yielded a single 249-base pair band, whereas TLR4 +896 G yielded 2 226- and 23-base pair bands.

Analysis for the polymorphism at codon 54 of exon I in the mannose-binding lectin gene was as described.²⁴ BanI digestion of the polymerase chain reaction product yielded 2 260- and 89-base pair bands (mannose-binding lectin +54 G) or a single uncut 349-base pair band (mannose-binding lectin + 54 A).

Analysis for the +1267 polymorphism, which contains a PstI restriction site in the inducible 70-kDa heat shock protein gene, was as described.²⁵ The 70-kDa heat shock protein +1267 A remained uncut and yielded a single 383-base pair band, whereas the 70-kDa heat shock protein +1267 G allele was cut into 2 244- and 139-base pair bands.

Genotype and allele frequencies were determined by direct counting and then dividing by the number of chromosomes to obtain allele frequency and by the number of women to obtain genotype frequency. The ² or Fisher exact tests were performed to determine whether there were differences in the frequencies of genotypes or alleles among the 3 groups of women. If a significant difference was found, a set of Bonferroni-adjusted pair-wise multiple comparisons (using = .05/3 = .017) was performed to determine differences between each group. Hardy–Weinberg equilibrium was determined by using a ² goodness of fit test based on expected frequencies. The expected frequencies were calculated using the assumption of Hardy–Weinberg equilibrium. There was no hospital-to-hospital variation in the results.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
A summary of the 8 gene polymorphisms examined and the consequences of the genetic alterations is outlined in Table 1. The alleles and genotypes of the polymorphic cytokine genes for each of the 3 ethnic groups studied are shown in Table 2. All were in Hardy–Weinberg equilibrium. No ethnic differences in the allelic frequencies of TNF--308 G>A, IL-1ß +3953 C>G, and IL-10 –1082 A>G genes were noted. In contrast, marked differences in the frequencies of IL-1ra and IL-4 gene alleles were observed. For example, IL1RN*1 homozygosity was more prevalent in blacks than in whites (P < .001) or Hispanics (P < .001). The IL1RN*2 allele was much less common in blacks than in whites (P < .001) or Hispanics (P < .001). In the IL-4 gene, the homozygous IL4 –590 C genotype was less common, and the IL4 –590 T homozygous genotype was more common, in blacks than in whites or Hispanics (P < .001). The frequency of the IL4 –590 T allele was highest in blacks followed by whites and Hispanics The differences between the frequencies of the IL4 –590 T allele for all ethnic groups were statistically significant (P < .001).

The alleles and genotypes of the polymorphic innate immune system genes for the 3 ethnic groups are shown in Table 3. Only the 70-kDa heat shock protein genotypes deviated from Hardy–Weinberg equilibrium (P <.001, P < .001, and P = .009 for whites, blacks, and Hispanics, respectively). No differences in toll-like receptor-4 allele frequencies in relation to ethnicity were observed. MBL +54G homozygosity was more common in the Hispanic women than in the white (P = .007) or black (P = .02) women. However, the difference between the frequency of the mannose-binding lectin +54G allele in Hispanics and the other ethnic groups did not reach statistical significance. Striking differences were noted, however, in the distribution of the 70-kDa heat shock protein genotype in relation to ethnicity. The 70-kDa heat shock protein +1267 A homozygosity was markedly reduced, and the 70-kDa heat shock protein + 1267 G homozygosity was markedly increased, in blacks as compared with whites (P < .001) or Hispanics (P < .002). The frequency of the 70-kDa heat shock protein +1267 G variant was higher in black women than in white (P < .001) and Hispanic (P = .002) women. Although the TNF- and 70-kDa heat shock protein genes are both located on the same chromosome, no linkage disequilibrium was observed between the 70-kDa heat shock protein +1267 and TNF- –308 loci (data not shown).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In our study population residing in New York City and Boston, black pregnant women strikingly differed from white and Hispanic pregnant women in the carriage of IL-1RN, IL-4–590, and 70-kDa heat shock protein +1267 alleles. Hispanic women also differed from whites in their carriage of the IL-4 –590T allele and the mannose-binding lectin +54G homozygous genotype. Such genomic variation observed between groups of individuals may partially explain ethnic differences in rates of specific pregnancy outcomes after exposure to inciting stimuli such as intraamniotic microbial infiltration.

The women in the present study all had uneventful pregnancy outcomes regardless of their genotypes. Possession of a specific genetic polymorphism is only a predisposing factor and does not by itself result in a pathological condition. Only after its activation may variations in the quantity, stability, and/or activity of the protein product of the polymorphic gene differentially influence the likelihood of a pathogenic response. Most likely only in the presence of microorganisms, microbial products, or nonmicrobial inducers of proinflammatory cytokine activity would the possession of certain alleles of the genes examined influence the risk for adverse pregnancy outcome. Thus, the magnitude of these genetic influences on pregnancy outcome will vary depending on exposure to eliciting stimuli.

The IL1RN genotype has been associated with the regulation of both IL-1ß and IL-1ra production,²⁶ but contradictory results have been reported. Interleukin-1–RN*2-positive mononuclear cells produced the highest levels of IL-1ß after in vitro stimulation.²⁶ Fetal carriage of IL1RN*2 has been shown to result in increased levels of both IL-1ß and IL-1ra in second-trimester amniotic fluid and in an elevated ratio of IL-1ß to IL-1ra.²⁷ Among pregnant women, carriage of this allele was associated with a greater vaginal concentration of IL-1ra and an increased isolation rate of Ureaplasma urealyticum.²⁸ Consistent with these observations are the associations between IL1RN*2 carriage and a variety of chronic inflammatory disorders (summarized elsewhere²⁹). Conversely, reduced IL-1 production by human monocytes carrying IL1RN*2 has been reported in 2 studies.^30,31 Similarly, maternal IL1RN*2 carriage during pregnancy was associated with a blunted IL-1ß response to anaerobic gram-negative rods and Gardnerella vaginalis colonizing the vagina (Genc MR, Onderdonk A, Vardhana S, Delaney ML, Norwitz ER, Tuomala RE, et al Polymorphism in intron 2 of interleukin 1 receptor antagonist gene, abnormal vaginal microflora in midtrimester, and preterm delivery [abstract]. Am J Obstet Gynecol 2003;189:32). In the present study, IL1RN*2 was less prevalent in blacks than in whites and Hispanics. Further studies are required to determine whether such ethnic/racial variations in maternal IL1RN*2 carriage may explain the ethnic disparity observed for certain adverse pregnancy outcomes. Additional investigations also are required to assess whether IL1RN*2 is in linkage disequilibrium with as-yet unidentified loci within the IL-1 gene complex that may differentially regulates IL-1ß production in response to specific environmental stimuli.

Like IL1RN, ethnic disparity was observed in the frequency of the IL-4 –590 T allele; that is, its frequency had the highest rate in blacks, followed by Hispanics, and then whites. IL4 –590 T was previously found to be higher among blacks than in whites in a Brazilian population with allelic frequencies similar to those observed in this study.³² Carriage of IL4 –590 T has been associated with elevated IL-4 production and increased susceptibility to atopy and asthma.³³ Because IL-4 is a prime inhibitor of proinflammatory cytokine production and cell-mediated immunity, elevated IL-4 production may increase susceptibility to infections complicating pregnancy. We speculate that the observed ethnic disparity in allelic frequencies for the IL-4 –590 C>T polymorphism may contribute to differences in the prevalence to infection-related preterm birth observed in different ethnic populations.

We observed no differences in allelic frequencies for the TNF –308 G>A, IL-1ß +3953 C>G, IL-10 –1082 A>G, and toll-like receptor-4 +896 A>G polymorphisms between whites, blacks, and Hispanics. Similarly, other investigators found no differences in allelic frequencies for these TNF- –308,^8,34 IL-10 –1082,⁸ and toll-like receptor-4 +896¹³ loci between whites and blacks.

Although no significant differences in allele frequency were observed, homozygosity for the mannose-binding lectin +54G allele was more common in Hispanics than in whites and blacks. Mannose-binding lectin +54G carriage has been associated with protection against Mycobacterium tuberculosis infection.³⁵ It remains to be determined whether the increased rate of mannose-binding lectin +54G homozygosity in Hispanics is the result of an evolutionary adaptation aimed to increase defense against intracellular microbial infections in this ethnic group.

A selection bias and deviation from Hardy–Weinberg equilibrium was observed in the 70-kDa heat shock protein genotype distributions in all ethnic groups in the present study as well as in previous investigations.^36,37 In addition, marked ethnic differences were noted. An allelic frequency of 66.4% for the 70-kDa heat shock protein +1267 G observed among blacks was significantly higher than that observed among whites and Hispanics. The findings were similar to a rate of 61.0% observed among African-American blacks in a previous study.³⁸ The allelic frequency of 70-kDa heat shock protein +1267 G in our nonblack subjects was also similar to that reported for Asians^25,39 and American whites.⁴⁰ The 70-kDa heat shock protein gene, located on chromosome 6 in the major histocompatibility complex (MHC) class III region,¹⁸ is one of the most highly conserved genes in evolution. The MHC region contains many genes with immunologic functions, and ethnic-specific linkage disequilibrium between an 70-kDa heat shock protein allele and polymorphisms in other MHC loci have been reported. The 70-kDa heat shock protein +1267G allele has been shown to have a linkage disequilibrium with DR2, and 70-kDa heat shock protein +1267A has a linkage disequilibrium with DR3 in whites^41,42 but not in blacks.³⁸ It remains to be determined whether a variable capacity to produce sufficient quantities of the 70-kDa heat shock protein in response to stressful stimuli or whether the linkage disequilibrium between the 70-kDa heat shock protein alleles and an unknown MHC gene is the primary factor accounting for the observed deviations from Hardy–Weinberg equilibrium. It is interesting to speculate that the relative infrequency of 70-kDa heat shock protein +1267A in black women may indicate a reduced resistance to stressful stimuli and an elevated risk for stress-related preterm birth in this ethnic group. Qualitative and quantitative variations in the stress response in pregnant women of differing ethnicity are currently being investigated.

	Footnotes

 
Supported in part by National Institutes of Health grants HD 41676, HD 35667, and M01RR0047 from the Division of Clinical Research, National Center for Research Resources. Daniel P. Nguyen was supported by a grant for young researchers from the University of Lausanne, Bourses de Perfectionnement et de Recherche, Switzerland.

Received March 9, 2004. Received in revised form April 23, 2004. Accepted April 29, 2004.

10.1097/01.AOG.0000133486.85400.5e

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