Genetic Variants and Susceptibility to Diseases of Prematurity in Very Low Birth-Weight Infants

Chronic lung disease of prematurity (CLD) is diagnosed in 30-40% of very low birth weight
(VLBW) infants (<1500gms) and remains a leading cause of mortality and long-term morbidity in
this population. Other diseases such as necrotizing enterocolitis (NEC), sepsis, patent
ductus arteriosus (PDA), and intraventricular hemorrhage (IVH) also contribute to mortality
and morbidity in this population. A central tenet in the pathogenesis of NEC, CLD and sepsis
is the failure of host genome-regulated immune defenses to surmount the challenges posed by
microbial pathogens in the presence of risk-factors that induce intestinal, pulmonary and
systemic injury, respectively. Toll like receptors (TLRs) are pathogen recognition receptors
which serve as the recognition and effector arm of the innate immune system. Since the
premature infant is predominantly dependent on the innate immune system for host defense our
hypothesis is that hypomorphic genetic variations in TLRs will increase susceptibility to
diseases of prematurity such as CLD and sepsis.The increased risk of inflammation, decreased
potential for repair and growth of lung may be reflected in the levels of biomarkers, such
as, cytokines, micro-RNAs (miRNA) or peptides that are secreted into biological fluids, like
blood and tracheal aspirates. It is likely that the genetic variants that increase the risk
of inflammation or decreased repair and growth will be also associated with altered levels of
these biomarkers. Measurement of the biomarkers and genetic variants together can increase
the precision of the early predictors of these complications of prematurity. Measurement of
biomarkers in biological fluids can be done with a shorter turnaround time than gene
sequencing at the present time. Biomarkers can also provide longitudinal changes in the
severity of disease and response to therapy. Newer multiplex assays make it feasible to
measure the levels of biomarkers in very small quantities of biological fluids. These
complementary strengths make gene sequencing and biomarker detection ideal 1-2 approach for
the early identification and disease progression evaluation in premature infants. Our
hypothesis will be tested in VLBW infants in four specific aims: (1) To determine whether the
presence of previously described single nucleotide polymorphisms (SNPs) in TLR4 (Asp299Gly,
Thr399Ile) TLR2 (Arg753Gln) and TLR5 (Arg392STOP) genes is associated with an increased risk
of CLD ; (2) to detect by DNA sequencing if novel genetic variations in TLR4, TLR2, TLR9,
MyD88 and other innate immune genes increases the risk of CLD or other disease in premature
infants; (3) to identify by gene sequence approach whether variants in other repair and
growth genes alter susceptibility to diseases affecting premature infants such as CLD,NEC or
sepsis or PDA or IVH; and (4) whether alterations in cytokines, miRNA and other biomarker
levels in serum from the same blood samples can provide additional predictive value in
recognizing the risk of diseases of prematurity. VLBW infants who develop CLD (oxygen
requirement at 36 weeks postconceptional age), NEC, sepsis, PDA, or IVH will serve as cases
while VLBW infants who do not develop the diseases of interest will serve as controls. A
0.5cc sample of blood will be collected from enrolled infants via indwelling catheter or
heel-stick for DNA analysis after consent is obtained. The TLR4 (Asp299Gly, Thr399Ile) TLR2
(Arg753Gln) and TLR5 (Arg392STOP) SNPs will be evaluated using a multiplex Single Base
extension based technique, using commercially available primers. Novel genetic variations in
these genes of interest and other variants proposed to be related to NEC will be detected
using conventional Sanger or Next Generation DNA sequencing. Serum will be separated from the
same blood sample obtained for DNA isolation. A second 0.5cc sample of blood will be
collected for biomarker measurement. Biomarker levels in serum will be analyzed using
multiplex EIA for cytokines, sequencing for miRNA profile and ELISA for peptide levels of
interest (VEGF, angiopoietin-2 and Nogo-B peptide). Benefits include the possibility of
development of risk-based preventive and therapeutic strategies to prevent CLD, NEC, sepsis,
PDA, or IVH in this population.

Inclusion Criteria:

- Infants born weighing less than 1500 grams

Exclusion Criteria:

- Infants born with congenital heart disease (other than patent ductus arteriosus)

- major congenital anomalies of the GI tract, respiratory tract, or kidneys