Microbiota, Necrotizing Enterocolitis And Genetic variations Of Zinc Transporter 2 In Preterm Infants

Open Access
Hamidi, Maryam
Graduate Program:
Public Health Sciences
Master of Science
Document Type:
Master Thesis
Date of Defense:
Committee Members:
  • Shannon Leanne Kelleher, Thesis Advisor
  • Maryam
  • Final
  • Thesis
  • NEC
  • project
ABSTRACT Necrotizing Enterocolitis (NEC) is a surgical emergency in Neonatal Intensive Care Units (NICUs) affecting 2-20% of preterm infants. In 2013, the incidence of NEC in the NICU at Penn State Hershey Medical Center was 18%. NEC is characterized by inflammatory damage to the intestinal tissues. The mortality of NEC is estimated to be between 15-20 % of infants diagnosed with the disease. Additionally, the majority of NEC survivors suffer from long-term complications, such as short bowel syndrome and neurodevelopmental delay. Combinations of different diagnostic tests are used to identify NEC; however, these tests are ordered when NEC is already clinically significant. Nearly 2/3 of infants diagnosed with NEC will need surgical interventions including laparatomy, bowel resection and ostomy establishment. These interventions have their own specific complications such as short bowel syndrome and long-term use of total parenteral nutrition. This represents a huge financial burden on the health care system. Given the acute onset and rapid progression of NEC, a non-invasive predictive method to identify risk factors that predispose premature infants to NEC will facilitate the development of interventions prior to the presentation of disease. Such a predictive method will be critical in reducing morbidity and mortality. Recently, Paneth cell (PC) dysfunction has been implicated in the pathogenesis of NEC. PCs are highly specialized and granulated secretory cells, which reside at the base of the small intestinal crypts. PC granules contain antimicrobial agents, which are critical for microbial defense, and provide a host-friendly environment in the small intestine. PC granules also contain a large amount of zinc (Zn). Granular Zn prevents degradation of antimicrobial proteins such as α-defensins and stabilizes lysozyme in its monomeric, active conformation promoting appropriate microbial defense. More than 30 years ago Santulli et al. discovered the relationship between intestinal bacterial communities and NEC. Pneumatosis intestinalis, a radiographic finding in many cases of NEC, is the result of intestinal microbial dysbiosis, illustrating the role of the microbial community in NEC pathogenesis. Additionally, the presence of specific bacteria such as Klebsiella seems to be elevated in the bowel before NEC development. Our unpublished data indicate that the Zn transporter ZnT2 (SLC30A2), may be critical to Zn accumulation in PC granules and important for optimal PC function, as ZnT2 knock-out mice have defects in PC granule Zn accumulation, secretion and antimicrobial activity. We recently identified genetic variants in ZnT2, resulting in loss of function [2] and thus, these individuals may be at increased risk for enteric infections and inflammatory diseases that require coordinated secretion of PC granules (Podany et al, Submitted manuscript). Many of these variants occur in exons 2, 3 and 7, and affect ZnT2 function and Zn secretion. In addition, numerous variants (A28D, K66N, Q71H, D103E, A105P, Q137H, T288S and T312K) display changes in the management sub-cellular Zn pools and Zn secretion causing “loss of function” and “gain of function” in cultured cells. Interestingly, 2 variants (D103E and T288S) were found in ~12% of the study population and had a strong impact on mammary cell function suggesting that these defects are profound enough to influence the function of a secretory cell. The objectives of this study were to determine if 1) variants in ZnT2 in premature infants are associated with alterations in gut microflora; and 2) if there is an association between variants of ZnT2 and the risk for NEC in premature infants. Our central hypothesis is that variants which result in the loss of ZnT2 function are associated with altered gut microbiota and NEC pathogenesis. In order to test this hypothesis, 51 preterm infants between the gestational ages of 26-36 weeks were recruited from the NICU at Penn State Hershey Medical Center within 48 hours after birth. DNA was collected by sterile buccal swab and the coding exons of SLC30A2 were sequenced. In addition, stool was collected with a sterile applicator two weeks after feeding was initiated. Fecal microflora was analyzed by 16S DNA sequencing. Data on feeding, birth weight, growth, race, ethnicity, gestational age, use of antibiotics, length of stay, mode of delivery, NEC diagnosis and gender were collected. Results indicated that there is an association between variants of ZnT2 and intestinal bacterial colonization in preterm infants. The relative abundance of Enterobactericeae is increased in infants with C-terminal and/or multiple variations of ZnT2. However, only one case of NEC was identified in our study. Overall, our study suggests that variants in ZnT2 create a less diverse microbial environment which may lead to short- or long-term health consequences.