Variant TLR4 nucleic acid and uses thereof

Details Variant TLR4 nucleic acid and uses thereof Variant TLR4 nucleic acid and uses thereof

1999-06-10

2004-05-25

Kunz, Gary (Department: 1647)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S023500, C536S024310, C536S024330, C424S009100

Reexamination Certificate

active

06740487

ABSTRACT:

BACKGROUND OF THE INVENTION
Endotoxin or lipopolysaccharide (LPS), released from the cell wall of bacteria, plays a central role in a broad spectrum of human disease. The pathogenic importance of LPS in gram-negative sepsis is well established. Intravenous LPS induces all of the clinical features of gram-negative sepsis, including fever, shock, leukopenia followed by leukocytosis, and disseminated intravascular coagulation (Favorite et al., 1942). Higher concentrations of circulating levels of endotoxin have been associated with manifestations of systemic inflammatory response syndrome (Wang et al., 1995) and the development of acute respiratory distress syndrome following sepsis (Brigham et al., 1986). Inhaled endotoxin can induce airflow obstruction in naive or previously unexposed subjects (Michel et al., 1992) and is the most important occupational exposure associated with the development Schwartz et al., 1995a), and progression (Schwartz et al., 1995b), of airway disease among exposed workers. The concentration of endotoxin in the domestic setting appears to be associated with the clinical severity of asthma (Michel et al., 1996). Moreover, recent studies have shown that endotoxin is a contaminant of particulate matter in air pollution and may play a role in the pathophysiologic consequences of air pollution (Bonner et al., 1998). Thus, endotoxin is an important cause of morbidity and mortality.
The ability of the host to respond to endotoxin may play an important role in determining the severity of the physiologic and biologic response to this frequently encountered toxin. In mice, genetic differences in susceptibility to LPS have been established. LPS hyporesponsiveness arose spontaneously and was first identified in the C3H/HeJ strain. This strain had an LD
50
for LPS at least 20 times that observed in A/HeJ mice (Sultzer et al., 1968). In addition to C3H/HeJ, two other mouse strains, C57BL10/ScCR (Coutinho et al., 1978) and its progenitor strain, C57BL/10ScN, (Vogel et al., 1979), are hyporesponsive to LPS.
Moreover, several reports suggest that humans may also respond differently to LPS. A patient with recurrent bacterial infections has been reported to be refractory to the in vivo and in vitro effects of LPS (Kuhns et al., 1997). Following challenge with intravenous LPS, this patient had no systemic increase in IL6 or G-CSF and had a minimal rise in the concentration of leukocytes, TNF-&agr;, and IL-8. Inter-individual differences have also been reported in the release and synthesis of cytokines by monocytes stimulated with LPS in vitro (Santamaria et al., 1989).
LPS is thought to cause much of its morbidity and mortality by activating kinases (DeFranco et al., 1998) that control the function of transcription factors (nuclear factor-&ugr;B and AP-1) and ultimately lead to production of pro-inflammatory cytokines and co-stimulatory molecules (Wright, 1999). Several lines of evidence suggest that the toll receptor (TLR) family, and specifically TLR4 and TLR2 regulate the interaction between LPS and intracellular kinases and may serve as a proximal target to interrupt LPS signaling (Wright, 1998; Medzhitov et al., 1997). Both TLR4 and TLR2 activate signaling through NF-&ugr;B and AP-1 in transfected human cell lines (Medzhitov et al., 1997; Yang et al., 1998), and TLR4 mediates LPS induced signal transduction (Chow et al., 1999). CD14, a glycosylphosphatidyl inositol-linked receptor that binds LPS (Poltorak et al., 1998a) enhances LPS induced TLR2 (Yanget al., 1998) and TLR4 (Chow et al., 1998) signaling, suggesting that the toll receptors interact with CD14 to initiate the cellular response to LPS. Studies in mice indicate that 1) the TLR4 gene maps to the critical region in LPS hyporesponsive mice (Poltorak et al., 1998), 2) mutations in the TLR4 gene (Poltorak et al., 1998; Qureshi et al., 1999) are found in mouse strains (C3H/HeJ and C57BL10/ScCr) that are defective in their response to LPS, and 3) disruption of the TLR4 gene results in a LPS hyporesponsive phenotype (Hoshino et al., 1999).
Thus, there is need to determine whether the human TLR4 gene is polymorphic, and whether any particular polymorphism is associated with disease, e.g., LPS hyporesponsiveness.
SUMMARY OF THE INVENTION
The invention provides a method to identify a mammal, e.g., a human, at risk of, or having, an indication associated with altered innate immunity, e.g., to bacterial infection. The method comprises contacting an amount of DNA obtained from a human physiological sample with an amount of at least one TLR4-specific oligonucleotide under conditions effective to amplify the DNA so as to yield amplified DNA. Then it is determined whether the amplified DNA comprises a nucleotide substitution, e.g., one that results in an amino acid substitution, i.e., the TLR4 DNA of the human encodes a variant TLR4. Thus, the invention is useful to detect polymorphisms in the TLR4 gene.
Normal healthy, non-asthmatic subjects demonstrate a reproducible airway response to an incremental LPS inhalation challenge test, with some subjects developing airflow obstruction when challenged with low concentrations of LPS and others virtually unaffected by high concentrations of inhaled LPS. These findings suggest that the spectrum of LPS responsiveness in humans is quite variable from one individual to the next (but reproducible within an individual), and that a substantial portion of the population may be hyporesponsive to inhaled LPS. As described hereinbleow, an incremental LPS inhalation challenge test was employed to reliably phenotype individuals as either responsive (at least a 20% decline in the forced expiratory volume in one second (FEV,) after inhaling up to 41.5 &mgr;g LPS) or hyporesponsive (FEV
1
, >80% of their baseline after inhaling 41.5 &mgr;g of LPS) to inhaled LPS. Fifty-two (63%) of these individuals were responsive to inhaled LPS and 31 (37%) were hyporesponsive to inhaled LPS.
These results were employed to determine the relationship between polymorphisms in the TLR4 gene and the airway response to inhaled LPS in the 83 normal healthy, non-asthmatic subjects. Using single standed conformational variant (SSCV) analysis and direct sequencing, a missense mutation (A896G) was identified in the fourth exon of the TLR4 gene that results in replacement of a conserved aspartic acid residue with glycine at position 299 in the extracellular domain of the TLR4 receptor. The Asp299Gly sequence variant occurred in 3 LPS responsive (5.8%) and 7 LPS hyporesponsive (22.6%) study subjects (p=0.03). Among the subjects with the common TLR4 allele (N=73), the dose-response slope (percent decline FEV
1
/cumulative dose of inhaled LPS) averaged a 1.86% decline in FEV
1
/&mgr;g inhaled LPS (range 0.01%-19.78%), while the dose-response slope for the subjects with the Asp299Gly allele (N=10) was significantly less (p=0.007), averaging 0.59% decline in FEV
1
/&mgr;g inhaled LPS (range 0.00%-1.59%). Thus, a sequence; polymorphism in the TLR4 gene, i.e., a missense mutation (Asp299Gly) in the fourth exon of the TLR4 gene, occurs in a substantial portion of the population, add frequency in other population Iowa, French and is associated with an airway hyporesponsive in humans challenged with inhaled LPS. The allelic frequency of the A896G substitution was 6.6% in the study population, 7.9% in a normal control population from Iowa (Lidral et al., 1998), and 3.3% in the parental chromosomes in of the CEPH population (NIH-CEPH, 1992).
The invention also provides an isolated and purified nucleic acid molecule comprising a nucleic acid segment, e.g., genomic DNA or cDNA, encoding TLR4, such as a variant TLR4. Also provided are primers, oligonucleotides and probes comprising the isolated nucleic acid sequences of the invention. The nucleic acid molecules of the invention may be single stranded or double stranded.
Transfection of CHO cells with either the wild-type or the mutant (Asp299Gly) allele of the TLR4 gene demonstrated that this mutation interrupts TLR4-mediated LPS signaling. Moreover, t

Affiliated with

Also associated with

Rating

Variant TLR4 nucleic acid and uses thereof does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Variant TLR4 nucleic acid and uses thereof, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Variant TLR4 nucleic acid and uses thereof will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3271237