Molecular Human Reproduction, Vol. 8, No. 11, 1031-1034,
November 2002
© 2002 European Society of Human Reproduction and Embryology
Implantation and pregnancy |
The CARD15 2936insC mutation and TLR4 896 A>G polymorphism in African Americans and risk of preterm premature rupture of membranes (PPROM)
1 Center for Research on Reproduction and Women’s Health and 2 Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania, Philadelphia, PA 19104 and 3 Perinatology Research Branch, National Institutes of Child Health and Human Development, Hutzel Hospital, Detroit, MI 48201, USA
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Infection is believed to be a leading cause of preterm premature rupture of membranes (PPROM). The bacterial cell wall component, lipopolysaccharide (LPS), is thought to initiate tissue responses leading to PPROM in the setting of Gram negative infection. LPS is recognized by the innate immune system, including the proteins encoded by the CARD15 and TLR4 genes. A recently described mutation (2936insC) in CARD15 and a polymorphism in TLR4 896 A>G impair responses to LPS. The objective of this study was to determine if African Americans, who have a higher incidence of PPROM than Caucasians, have different frequencies of the mutant CARD15 allele and the TLR4 hyporesponsive variant, and if risk of PPROM is influenced by fetal carriage of these alleles. The allele frequencies for the CARD15 mutation and the TLR4 896G variant in African Americans were similar to those reported for Caucasians. There was no association between the TLR4 alleles examined and PPROM. However, the CARD15 mutation was only detected in controls and not in PPROM cases. We conclude that the CARD15 mutation and hyporesponsive TLR4 allele do not contribute to ethnic variation in the incidence of PPROM.
/gene mutation/polymorphism/PPROM/TLR4
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Intrauterine infection has been implicated in the aetiology of preterm premature rupture of membranes (PPROM), which accounts for 30–40% of all preterm deliveries (Parry and Strauss, 1998
). Lipopolysaccharide (LPS), a component of the cell wall of Gram negative bacteria, is thought to play a key role in eliciting an inflammatory response including the activation of the immune cells and the release of enzymes involved in remodelling of the extracellular matrix leading to PPROM (Romero et al., 1987Romero et al., 1988). It has recently been discovered that LPS is recognized by proteins of the innate immune system, including Toll-like receptor 4 (TLR4) (Medzhitov et al., 1997) and CARD15, also referred to as Nod2 (Ogura et al., 2001a,b).
CARD15, a member of the NOD1/APAF1 gene family, is a cytoplasmic protein homologous to plant disease resistance proteins. It has leucine-rich repeats that are thought to be required for LPS recognition located in its C-terminus as well as two N-terminal caspase recruitment domains, implicating the protein in programmed cell death (Beutler, 2001). CARD15 is thought to have a role in inflammatory responses to microbial products, especially LPS, through activation of a signal transduction system involving NF-kB (Inohara and Nunez, 2001; Ogura et al., 2001a). This signal transduction pathway can result in the increased expression of pro-inflammatory cytokines and matrix metalloproteinases, which have been associated with PPROM and are thought to be central to the untimely rupture of the fetal membranes (Parry and Strauss, 1998).
Several mutations have been identified in the human CARD15 gene. An insertion mutation causing a frameshift, 2936insC, results in a truncated protein missing the 33 C-terminal amino acids in the leucine-rich repeat domain. The truncated CARD15 protein cannot efficiently activate NF-kB in response to LPS (Ogura et al., 2001a). The CARD15 insertion mutation, as well as other CARD15 mutations, have been linked to familial Crohn’s disease and Blau syndrome, chronic inflammatory disorders (Hugot et al., 2001; Miceli-Richard et al., 2001; Ogura et al., 2001a). Thus, CARD15 has seemingly paradoxical activities, apparently triggering a cellular response to LPS on the one hand, but evidently suppressing the chronic inflammatory reaction to pathogens on the other.
TLR4 is a transmembrane protein also containing repeated leucine-rich motifs in its extracellular portion. TLR proteins also contain a cytoplasmic domain that is homologous to the interleukin-1 receptor that triggers NF-kB signalling pathways (Medzhitov et al., 1997). TLR4 is expressed on cells of the immune system, and its activation in macrophages causes the release of cytokines and the expression of co-stimulatory molecules that are essential for activating cells of the adaptive immune system (Modlin et al., 1999). Hyporesponse to a LPS challenge has been associated with a TLR4 variant resulting in an amino acid substitution (Asp299Gly) lying between leucine-rich repeats (Arbour et al., 2000).
We hypothesized that mutations in genes encoding the innate immune system proteins responsible for recognition/induction of signalling of LPS might affect the risk of PPROM, based on the observation that infection is linked to PPROM and LPS is thought to be a trigger for tissue remodelling associated with Gram negative infection. To test this hypothesis, we determined the frequency of the CARD15 mutant and TLR4 variant alleles in African Americans, who have an 
2-fold greater risk of PPROM than Caucasians (Schieve and Handler, 1996; Martin et al., 2001). Moreover, chorioamniotis or the presence of amniotic fluid markers indicating infection is more prevalent in African Americans (Guinn et al., 1995; Fiscella, 1996). We also conducted a case-control study on African Americans to determine if carriage of the CARD15 2936insC mutation or hyporesponsive TLR4 allele is protective against PPROM. Because our recent work on polymorphisms in genes encoding matrix metalloproteinases suggested that the fetus contributes significantly to the risk of PPROM (Ferrand et al., 2002; Fujimoto et al., 2002), the association study was focussed on fetal CARD15 and TLR4 allelic variation.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Subjects
Subjects in this study were offspring of African American women receiving obstetrical care at the Hospital of the University of Pennsylvania, Philadelphia, PA and Hutzel Hospital, Detroit, MI, USA. The University of Pennsylvania, Wayne State University and National Institute of Child Health and Human Development Institutional Review Boards approved the collection of samples for genetic studies. Subjects were recruited sequentially between January 1997 and January 2002. Informed written consent was obtained from mothers prior to collection of biological material including specimens for extraction of DNA. Cases (n = 131) were African American neonates from pregnancies complicated by spontaneous rupture of membranes prior to onset of labour before 37 weeks of completed gestation. The diagnosis of membrane rupture was based on pooling of vaginal fluid and a positive nitrazine test. Control subjects were also recruited sequentially over the same time period (n = 246 African Americans and 58 Caucasians). The control neonates were from normal pregnancies delivered at term from mothers with no prior history of PROM/PPROM or preterm birth. Patients with multiple gestations, fetal anomalies, and medical complications of pregnancy requiring induction of labour were excluded. Although attempts were made to recruit all eligible subjects seen during this time period some were lost to follow-up, some women refused to participate in the study and in a minority of cases a fetal DNA sample was not obtained. Enrollment of eligible subjects approached 75% for cases and 60% for controls. DNA was not available for analysis of both genes in some instances or PCR amplification consistently failed, precluding genotyping. This accounts for differences in numbers of subjects analysed in the association studies.
Analysis of CARD15 and TLR4 alleles
DNA was extracted from umbilical cords, cord blood, or neonate cheek swabs by conventional methods as described previously (Ferrand et al., 2002). We extracted DNA from bowel resected from three subjects with Crohn’s disease in order to obtain control samples that were likely to be heterozygous or homozygous for the CARD15 insertion mutation. Two of the subjects were homozygous for the mutation, one was heterozygous.
For the 2936insC mutation, we amplified a region of the CARD15 gene using a forward primer 5'-CTGAGCCTTTGTTGATGAGC-3' and a reverse primer 5'-CCTTACCAGACTTCCAGGATG-3'. PCR was carried out using Ready-To-Go PCR Beads (Amersham Pharmacia Biotech Inc.). After initial denaturation at 94°C for 5 min, PCR was performed for 35 cycles of denaturation at 94°C for 1 min, annealing at 64°C for 30 s, and extension for 1 min at 72°C. The 258 bp reaction products containing the 2936insC region were digested with restriction endonuclease Nla IV (New England Biolabs) and fractionated on a 3% agarose gel. This enzyme cuts the sequence GGNNCC, recognising PCR products that have the 2936 C insertion. We sequenced 35 samples to confirm that the restriction enzyme digestion method accurately identified the CARD15 insertion mutation.
To detect TLR4 896 A>G polymorphism, we used the PCR amplification strategy previously described (Lorenz et al.., 2001), employing mismatch primers designed to detect the wild type and variant TLR 4 alleles based on the presence of a restriction site in the variant allele. The following primers were used: forward 5'-GATTAGCATACTTAGACTACTACCTCCATG-3' and reverse 5'-GATCAACTTCTGAAAAAGCATTCCCAC-3'. The underlined base in the forward primer indicates the nucleotide altered to create a Nco I restriction site in the presence of the polymorphism. PCR was carried out using Platinum Taq (Gibco BRL). After initial denaturation at 94°C for 5 min, PCR was performed for 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension for 30 s at 72°C, after which a 20 µl sample was digested with Nco I (New England Biolabs) and fractionated on a 3% agarose gel.
Statistical analysis
Cases and controls were compared on demographic factors using Student’s t-test. Means and standard errors are reported for the data in Table II. Comparisons of proportions of allele frequencies were performed using the 
-2 test or Fisher’s exact test.
|
We estimated that our study had 80% power with an alpha of 0.05 to detect an odds ratio of 7.6 with respect to TLR4 mutations and protection against PPROM. Thus, we would fail to detect a smaller but significant contribution of TLR4 allelic variation to risk of PPROM.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
A new method based on diagnostic restriction enzyme digestion patterns of PCR products was designed to detect the 2936insC CARD15 mutation. This procedure reliably identified the different alleles (Figure 1
). The genotype determined by restriction digestion was concordant with that determined by DNA sequence analysis in 35 subjects. We were able to successfully genotype 96.9% of samples. Failed PCR amplification of genomic DNA was the reason for the inability to ascertain the genotype in the remaining 3.1%. The frequencies of the 2936insC mutation and the hyporesponsive TLR4 alleles in our African American population were similar to those previously reported for Caucasians and as confirmed by our analysis (P 
0.05 difference by Fisher’s exact test) (Table I). All of the carriers of the hyporesponsive alleles were heterozygotes in our African American and Caucasian populations.
|
|
To determine if there was an association between PPROM and the CARD15 insertion mutation or the hyporesponsive TLR4 allele, we performed a case-control study in which we genotyped fetal DNA from cases (PPROM) and controls. The demographic characteristics of our study population are presented in Table II
. The mothers were similar in age and parity, but there was a small but significant difference in gravidity, and as expected, significant differences in gestational age at birth and birth weight. There was no significant difference in the frequencies of the TLR4 alleles among cases and controls and all carriers of the G allele were heterozygotes. Interestingly, the 2936insC mutation was only detected in controls, all of whom were heterozygous for the mutation. The absence of this mutation in cases was statistically significant (P = 0.017; Table III) and would remain so even after Bonferroni correction for testing for allelic variants in two different genes. However, given the low frequency of the mutant CARD15 allele, it could make only a minor contribution to protection against PPROM.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Our findings indicate that the CARD15 insertion mutation and the hyporesponsive TLR4 allele are unlikely to contribute to ethnic differences in the incidence of PPROM among African Americans and Caucasians because they occur at similar frequencies in African Americans and Caucasians. This finding is consistent with epidemiological data suggesting that Crohn’s disease occurs as frequently in African Americans as Caucasians (Bartholomew and Butler, 1979
; Calkins et al., 1984) and is contrary to the notions of some that Crohn’s disease is less prevalent in African Americans when compared with Caucasians (Dinkel et al., 1986; Paul et al., 1990).
The association of CARD15 mutations with Crohn’s disease and Blau syndrome (Hugot et al., 2001; Miceli-Richard et al., 2001; Ogura et al., 2001) indicates that functional impairment of CARD15 results in a chronic inflammatory state. An overactive immune system in these conditions, perhaps because of a defect in programmed death of inflammatory cells resulting from defects in caspase activation (Beutler, 2001), might be expected to contribute to inflammatory changes in the fetal membranes and possibly increase, rather than reduce, the risk of PPROM. However, the relationship between the chronic inflammatory response of Crohn’s disease and Blau syndrome and the inflammatory reaction engendered by infection that leads to PPROM is not known. Maternal Crohn’s disease is associated with increased risk of preterm birth and low birth weight, reflecting the impact of either the underlying disease or the treatments (Baird et al., 1990; Fonager et al., 1998). The incidence of PPROM in pregnancies in which the fetus carries a CARD15 mutation has not been previously reported, nor has the incidence of PPROM in women with Crohn’s disease been systematically evaluated (Hannan and Kirsner, 1985; Katz and Pore, 2001).
If one assumes that PPROM can result from cellular responses to LPS, mutations that impair LPS activation of CARD15 in fetal membranes might be predicted to protect against PPROM. The fact that we did not detect the CARD15 2936insC mutation in PPROM cases is consistent with this notion. Although the statistical significance for the apparent protective effect of the 2936insC mutation was not high, the relatively small sample size in our study and the relatively low frequency of the mutation may have obscured a more striking effect. Furthermore, we did not determine the incidence of chorioamnionitis or bacterial vaginosis in our study population or correlate infection with CARD15 genotype and pregnancy outcome. Moreover, CARD15 activates NF-kB differentially in response to LPS from different organisms (Ogura et al., 2001b). It is possible that more significant protection against PPROM would have emerged if cases were selected using the criteria of PPROM with concomitant Gram negative infection by identified pathogens. This should be the subject of future investigation.
Because the results of association studies can be influenced by population stratification, which is a particular concern when heterogeneous ethnic groups such as African Americans are investigated, as well as by the history of the alleles in question in the population, our findings may not be generalized to other ethnic groups. It should also be recognized that association studies do not establish linkage of the gene being studied to the outcome. Other genes in linkage disequilibrium with the investigated alleles could account for the association. Recognizing these caveats, we conclude that there are no significant ethnic differences in the frequencies of the CARD15 2936insC mutation and the hyporesponsive TLR4 allele among African Americans and Caucasians. However, the fetal CARD15 insertion mutation impairing LPS responsiveness appears to be less frequent in pregnancies complicated by PPROM, possibly suggesting a protective effect of the mutant allele.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
We thank Ms Judith Wood for assistance in preparation of this manuscript. This research was supported by NIH grants HD34612 and D43/TWO1272 and a grant from the Bill and Melinda Gates Foundation. S.P. was supported by the Women’s Reproductive Health Research Career Development Center grant HDO1265.
|
Notes |
|---|
4 To whom correspondence should be addressed at 1354 BRB II, 421 Curie Boulevard, Philadelphia, PA 19104, USA. E-mail: jfs3{at}mail.med.upenn.edu
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Arbour, N.C., Lorenz, E., Schutte, B.C., Zabner, J., Kline, J.N., Jones, M., Frees, K., Watt, J.L. and Schwartz, D.A. (2000) TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nat. Genetics, 25, 187–191.[Web of Science][Medline]
Baird, D.D., Narendranathan, M. and Sandler, R.S. (1990) Increased risk of preterm birth for women with inflammatory bowel disease. Gastroenterology, 99, 987–994.[Web of Science][Medline]
Bartholomew, C. and Butler, A. (1979) Inflammatory bowel disease in the West Indies. Br. Med. J., ii, 824–825.
Beutler, B. (2001) Autoimmunity and apoptosis: The Crohn connection. Immunity, 15, 5–14.[Web of Science][Medline]
Calkins, B.M., Lilienfeld, A.M., Garland, C.F. and Mendeloff, A.I. (1984) Trends in incidence rates of ulcerative colitis and Crohn’s disease. Dig. Dis. Sci., 29, 913–920.[Web of Science][Medline]
Dinkel, E., Dittrich, M., Peters, H. and Baumann, W. (1986) Real-time ultrasound in Crohn’s disease: characteristic features and clinical implications. Pediatr. Radiol., 16, 8–12.[Web of Science][Medline]
Ferrand, P., Parry, S., Sammel, M., Macones, G.A., Kuivaniemi, H., Romero, R. and Strauss, J.F. III, (2002) A polymorphism in the matrix metalloproteinase 9 (MMP-9) promoter is associated with increased risk of preterm premature rupture of membranes (PPROM) in African Americans. Mol. Hum. Reprod., in press.
Fiscella, K. (1996) Race, perinatal outcome, and amniotic infection. Obstet. Gynecol. Survey, 51, 60–66.[Medline]
Fonager, K., Sorensen, H.T., Olsen, J., Dahlerup, J.F. and Rasmussen, S.N. (1998) Pregnancy outcome for women with Crohn’s disease: a follow-up study based on linkage between national registries. Am. J. Gastroenterol., 93, 2426–2430.[Web of Science][Medline]
Fujimoto, T., Parry, S., Urbanek, M., Sammel, M., Macones, G., Kuivaniemi, H., Romero, R. and Strauss, J.F. III (2002) A single nucleotide polymorphism in the matrix metalloproteinase-1 (MMP-1) promoter influences amnion cell MMP-1 expression and risk for preterm premature rupture of the fetal membranes. J. Biol. Chem., 277, 6296–6302.
Guinn, D.A., Goldenberg, R.L., Hauth, J.C., Andrews, W.W., Thom, E. and Romero, R. (1995) Risk factors for the development of preterm premature rupture of the membranes after arrest of labor. Am. J. Obstet. Gynecol., 173, 1310–1315.[Web of Science][Medline]
Hanan, I.M. and Kirsner, J.B. (1985) Inflammatory bowel disease in the pregnant woman. Clin. Perinatol., 12, 669–682.[Web of Science][Medline]
Hugot, J.P., Chamaillard, M., Zouali, H., Lesage, S., Cezard, J.P., Belaiche, J., Almer, S., Tysk, C., O’Morain, C.A., Gassull, M. et al. (2001) Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature, 411, 599–603.[Medline]
Inohara, N. and Nunez, G. (2001) The NOD: a signaling module that regulates apoptosis and host defense against pathogens. Oncogene, 20, 6473–6481[Web of Science][Medline]
Katz, J. A. and Pore, G. (2001) Inflammatory bowel disease and pregnancy. Inflamm. Bowel Dis., 7, 146–157.[Web of Science][Medline]
Lorenz, E., Frees, K.L.and Schwartz, D.A. (2001) Determination of the TLR4 genotype using allele-specific PCR. Biotechniques, 31, 22–24.[Web of Science][Medline]
Martin, J.A., Hamilton, B.E. and Ventura, S.J. (2001) Births: preliminary data for 2000. Natl Vital Stat. Rep., 49, 1–20.[Medline]
Medzhitov, R., Preston-Hurlburt, P. and Janeway, Jr., C.A. (1997) A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature, 388, 394–397.[Web of Science][Medline]
Miceli-Richard, C., Lesage, S., Rybojad, M., Prieur, A.M., Manouvrier-Hanus, Hafner, R., Chamaillard, M., Zouali, H., Thomas, G. and Hugot, J.P. (2001) CARD15 mutations in Blau syndrome. Nature Genet., 29, 19–20.[Web of Science][Medline]
Modlin, R.L., Brightbill, H.D. and Godowski, P.J. (1999) The toll of innate immunity on microbial pathogens. New Engl. J. Med., 340, 1834–1835.
Ogura, Y., Bonen, D. K., Inohara, N., Nicolae, D.L., Chan, F.F., Ramos, R., Britton, H., Maran, T., Karaliuskas, R., Duerr, R.H. et al. (2001b) A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature, 411, 603–606.[Medline]
Ogura, Y., Inohara, N., Benito, A., Chen, F.F., Yamoaka, S. and Nunez, G. (2001a) Nod2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates NF-kappaB. J. Biol. Chem., 276, 4812–4818.
Parry, S. and Strauss, J. F. III (1998) Premature rupture of the fetal membranes. N. Engl. J. Med., 338, 663–670.
Paul, H., Jr., Barnes, R. W., Reese, V. E., Childress, M.H., Scott, V. and Lefall, L.D. (1990) Crohn’s disease in black patients. J. Natl Med. Assoc., 82, 709–712.[Medline]
Romero, R., Kadar, N., Hobbins, J.C. and Duff, G.W. (1987) Infection and labor: The detection of endotoxin in amniotic fluid. Am. J. Obstet. Gynecol., 157, 815–819.[Web of Science][Medline]
Romero, R., Roslansky, P., Oyarzun, E., Wan, M., Emanian, M., Novitsky, T.J., Gould, M.J. and Hobbins, J.C. (1998) Labor and infection. II. Bacterial endotoxin in amniotic fluid and its relationship to the onset of preterm labor. Am. J. Obstet. Gynecol., 158, 1044–1049.
Schieve, L.A. and Handler, A (1996) Preterm delivery and perinatal death among Black and White infants in a Chicago-area perinatal registry. Obstet. Gynecol., 88, 356–363.[Web of Science][Medline]
Submitted on March 26, 2002; resubmitted on June 13, 2002; accepted on August 9, 2002.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  G. Rey, F. Skowronek, J. Alciaturi, J. Alonso, B. Bertoni, and R. Sapiro Toll receptor 4 Asp299Gly polymorphism and its association with preterm birth and premature rupture of membranes in a South American population Mol. Hum. Reprod., September 1, 2008; 14(9): 555 - 559. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A. Awomoyi, P. Rallabhandi, T. I. Pollin, E. Lorenz, M. B. Sztein, M. S. Boukhvalova, V. G. Hemming, J. C. G. Blanco, and S. N. Vogel Association of TLR4 Polymorphisms with Symptomatic Respiratory Syncytial Virus Infection in High-Risk Infants and Young Children J. Immunol., September 1, 2007; 179(5): 3171 - 3177. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. C. Barber, L.-Y. E. Chang, B. D. Arnoldo, G. F. Purdue, J. L. Hunt, J. W. Horton, and C. C. Aragaki Innate Immunity SNPs are Associated with Risk for Severe Sepsis after Burn Injury Clin. Med. Res., December 1, 2006; 4(4): 250 - 255. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R C Barber, C C Aragaki, F A Rivera-Chavez, G F Purdue, J L Hunt, and J W Horton TLR4 and TNF-{alpha} polymorphisms are associated with an increased risk for severe sepsis following burn injury J. Med. Genet., November 1, 2004; 41(11): 808 - 813. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Illig, F. Bongardt, A. Schopfer, R. Holle, S. Muller, W. Rathmann, W. Koenig, C. Meisinger, H.-E. Wichmann, and H. Kolb The Endotoxin Receptor TLR4 Polymorphism Is Not Associated With Diabetes or Components of the Metabolic Syndrome Diabetes, November 1, 2003; 52(11): 2861 - 2864. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||