Study of the Effect of SNPs in p53 and p53 Response Elements on the Inflammatory Response to DNA Damage

This research study will investigate the role of SNPs in p53 and p53 response elements on the
inflammatory response to DNA damage. A total of 200 participants aged 18 years and older
carrying one of the five SNPs of interest and wild-type controls will be identified and
recruited from the Environmental Polymorphism Registry (EPR). In addition, participants will
be recruited based on their health outcomes and SNP associations from the EPR registry to
study genotype-phenotype effects on lymphocytes. The EPR is a long-term project to collect
and store up to 15,000 DNA samples for use in research studies from individuals in the
greater North Carolina Triangle Region.

This observational gene association study will recruit participants on the basis of genotype
or phenotype and then observe the lymphocyte response to chemotherapeutic agents and relevant
environmental pathogens. The SNPs of interest are p53, as well as four of its downstream
target genes including FLT1, MDM2, TLR8 and RRM1. A maximum of 320 mLs of blood will be
obtained from each participant during one visit lasting approximately one hour. Cells from
the donated blood samples will be examined for their response to exposed environmental stress
ex vivo.

The primary objective is to determine the association between five SNPs and p53 target gene
expression after exposure to Nutlin or doxorubicin (chemotherapeutic agents) with outcome
measured by RT-PCR. The five SNPs are p53 rs1042522, MDM2 rs2279744, FLT1 C-677T, TLR8
rs3761624 and RMM1 rs1465952. The secondary objectives are to: (1) to determine the p53
promoter occupancy measured by ChIP analysis for the following SNPs: FLT1 C-677T, TLR8
rs3761624 and RMM1 rs1465952; (2) to measure apoptosis by Annexin V-PI assay for p53
rs1042522 SNPs; (3) to examine the cell cycle profile analysis (FACS) by cytofluorometry for
p53 rs1042522SNPs; and (4) to determine DNA repair using Pulse Field Electrophoresis Gel
(TAFE gels) for the following p53 rs1042522SNPs. Furthermore, the association between the
SNPs of interest and phenotypic characteristics will be explored using the EPR health and
exposure survey to identify significant genotype-phenotype associations in the EPR
population. The effect of the associations will be tested on lymphocyte function after
exposure to Nutlin or doxorubicin.

We have established that p53 can greatly alter expression of many immune genes including most
of the toll-like receptor (TLR) innate immunity genes which are considered important
components of antiviral immunity against HIV infection. Given the unique roles of TLR
signaling during acute HIV-1 infection and their potential role in chronic inflammation, it
is important to elucidate whether TLR polymorphisms contribute to HIV-1 pathogenesis and
variability in disease progression.

Recently, we confirmed that p53 can target the TLR8 ssRNA responsive receptor in a single
nucleotide polymorphism (SNP)-dependent manner (rs3761624). We have shown in a human study
that a SNP within a p53 response element of the TLR8 promoter can strongly influence
respiratory syncytial virus (RSV)-associated disease in infants. In addition, there are SNPs
in the coding region of p53 that alter the amplitude of signaling attributed to this protein.
Projecting these findings to HIV and AIDS, we hypothesize that p53 is a downstream effector
that initiates anti-proliferative innate immune responses to viruses and other pathogens,
establishing a new role for p53. These novel investigations will provide critical
understanding of the role of innate restriction factors in resistance to HIV-1 and disease
progression.

Overall, we hope the results of this study lead to discovery of important information
regarding the role of SNPs located in p53 and p53 response elements in human disease,
potentially identifying new targets for future studies.

- INCLUSION CRITERIA:

- Male or female 18 years of age or older

- Participants must be able to understand and provide written informed consent to
participate in the study

- Participants must be able to travel to the CRU

- Nonpregnant

- Healthy participants as defined by the International Red Cross guidelines (Healthy
means that an individual feels well and can perform normal activities. If the
individual has a chronic condition such as diabetes or high blood pressure, healthy
also means that they are being treated and the condition is under control).

- Participants with health outcomes identified by genotype-phenotype association
studies.

- HIV-1 seropositive under medicament treatment (for HIV P53 and TLR8 groups only)
(checked every 6 months at visit)

EXCLUSION CRITERIA:

- Use of immunosuppressants or other immune-modifying drugs [e.g., Rituxan, Humira,
Enbrel, Cyclosporin (Neoral, Sandimmune, and SangCya), Azathioprine (Imuran)],
Monoclonal antibodies [e.g., infliximab (Remicade)] (for healthy controls only)

- Chronic use of systemic, inhaled steroids

- Current or use of prescription strength topical corticosteroids within the last 7days

- History of cancer, including skin cancer (for healthy controls only)

- History of chemotherapy or radiation treatment (for healthy controls only)

- Confirmed or suspected immunosuppressive or immunodeficient condition (for healthy
controls only)

- Hepatitis B/C-positive status (checked every 6 months at visit)

- Body weight < 50 kg (<110 lbs)

- If blood donation exceeds 200ml:

--Hematocrit <34% for women or <36% for men, or >56% for either gender

- Temperature > 37.6 C; blood pressure < 90/50 mm Hg or blood pressure >160/100 mm Hg;
pulse rate < 50 or > 100 beats/minute

- Blood or plasma donation that will cause the participant to exceed 550ml of blood in
the last 8 weeks

- HIV1 donors with opportunistic infections:

- Candidiasis of bronchi, trachea, esophagus, or lungs

- Invasive cervical cancer

- Coccidioidomycosis

- Cryptococcosis

- Cryptosporidiosis, chronic intestinal (greater than 1 month's duration)

- Cytomegalovirus disease (particularly CMV retinitis)

- Encephalopathy, HIV-related

- Herpes simplex: chronic ulcer(s) (greater than 1 month's duration); or
bronchitis, pneumonitis, or esophagitis

- Histoplasmosis

- Isosporiasis, chronic intestinal (greater than 1 month's duration)

- Kaposi's sarcoma

- Lymphoma, multiple forms

- Mycobacterium avium complex

- Tuberculosis

- Pneumocystis carinii pneumonia

- Pneumonia, recurrent

- Progressive multifocal leukoencephalopathy

- Salmonella septicemia, recurrent

- Toxoplasmosis of brain

- Wasting syndrome due to HIV

- HIV and hepatitis results will be confidentially obtained. Testing will be
contracted to an external certified laboratory. Results will be available
only to the study team, with the few caveats that follow:

All positive HIV, hepatitis B, and hepatitis C results that are unexpected (i.e.
participant is not currently enrolled in the EPR as an HIV-1 seropositive under medicament
treatment) will be promptly communicated to the donor by the study doctor/PI or the CRU
Director. The participant will be referred to their physician and/or to the N.C. Department
of Health for confirmatory testing and counseling. As explained in detail in the attached
Supplement describing N.C. State Department of Health code will be followed. The state code
mandates reporting of positive results along with the participant s name and identifying
information to the N.C. Department of Public Health. Upon contracting with the testing
laboratory, clarification will be obtained and documented as to whether the contracted
laboratory or the study MD will be responsible for reporting positive results to the state
to avoid duplication of reporting. Upon receipt of the test results, the N.C. Department of
Health will contact the participant to inform them of the positive result, how to find
care, how to avoid infecting others, how the newly diagnosed HIV and/or hepatitis infection
is reported, and the importance of informing their partners at possible risk because of
their HIV and/or hepatitis infection. If the HIV, hepatitis B, and hepatitis C results are
negative, the participant will be not be notified. However, the participant may contact the
research study nurse for their results. HIV and hepatitis B/C test results, non-reactive
and reactive, will be documented confidentially by the PI or study coordinator in the
subject s file, and kept in a locked file

cabinet in the CRU Medical Records Room. In order to document the reporting procedure and
the time associated with the reporting process, a document has been created and placed in
the study specific manual (Hepatitis B/C and HIV Notification Process for Reactive Results
Form).

-Healthy control participants carrying SNPs TLR8 and FLT1 who are currently taking hormonal
contraception (e.g. oral contraceptives, IUDs with hormones, contraceptive patches) or
hormone replacement therapy will be excluded from the study unless the participant has been
off of the hormone treatment for 1 month or longer.