Genetic variation in environmental enteropathy and stunting in Zambian children: A pilot genome wide association study using the H3Africa chip.

PURPOSE: Stunting is known to be heavily influenced by environmental factors, so the genetic contribution has received little attention. Here we report an exploration of genetic influences in stunted Zambian children with environmental enteropathy. METHOD: Children with stunting (LAZ < -2) were enrolled and given nutritional therapy. Those that were non-responsive to therapy were designated as cases, and children with good growth (LAZ > -1) from the same community as controls. Blood and stool samples were taken to measure biomarkers of intestinal inflammation, epithelial damage, and microbial translocation. Single nucleotide polymorphism array genotyping was carried out on saliva samples using the H3Africa consortium array. RESULTS: Genome wide associations were analysed in 117 cases and 41 controls. While no significant associations with stunting were observed at P<5x10-8, likely due to the small sample size, interesting associations were observed at lower thresholds. SNPs associated with stunting were in genomic regions known to modulate neuronal differentiation and fatty acid biosynthesis. SNPs associated with increased microbial translocation were associated with non-integrin membrane ECM interactions, tight junctions, hemostasis, and G-alpha signalling events. SNPs associated with increased inflammation were associated with, ECM interactions, purine metabolism, axon guidance, and cell motility. SNPs negatively associated with inflammation overlapped genes involved in semaphoring interactions. We explored the existing coeliac disease risk HLA genotypes and found present: DQ2.5 (7.5%), DQ8 (3.5%) and DQ2.2 (3.8%); however, no children were positive for coeliac antibodies. We detected HLA-DRB:1301 and HLA-C:1802 with high odds ratios and P<0.05 in stunted children compared to controls. CONCLUSION: Genetic variations associated with stunting and the enteropathy underlying it, include variants associated with multiple pathways relating to gene expression, glycosylation, nerve signalling, and sensing of the nutritional and microbiological milieu.

Potential determinants of low circulating glucagon-like peptide 2 concentrations in Zambian children with non-responsive stunting.

NEW FINDINGS: What is the central question of this study? Non-responsive stunting is characterised by a progressive decline of circulating glucagon-like peptide 2: what are the possible causes of this decline? What is the main finding and its importance? In contrast with the established loss of Paneth and goblet cells in environmental enteropathy, there was no evidence of a parallel loss of enteroendocrine cells as seen by positive tissue staining for chromogranin A. Transcriptomic and genomic analyses showed evidence of genetic transcripts that could account for some of the variability seen in circulating glucagon-like peptide 2 values. ABSTRACT: Nutrient sensing determines digestive and hormonal responses following nutrient ingestion. We have previously reported decreased levels of glucagon-like peptide 2 (GLP-2) in children with stunting. Here we demonstrate the presence of enteroendocrine cells in stunted children and explore potential pathways that may be involved in reduced circulating levels of GLP-2. At the time of performing diagnostic endoscopies for non-responsive stunted children, intestinal biopsies were collected for immunofluorescence staining of enteroendocrine cells and transcriptomic analysis. Circulating levels of GLP-2 were also measured and correlated with transcriptomic data. An exploratory genome-wide association study (GWAS) was conducted on DNA samples (n = 158) to assess genetic contribution to GLP-2 variability. Intestinal tissue sections collected from non-responsive stunted children stained positive for chromogranin A (88/89), alongside G-protein-coupled receptors G-protein receptor 119 (75/87), free fatty acid receptor 3 (76/89) and taste 1 receptor 1 (39/45). Transcriptomic analysis found three pathways correlated with circulating GLP-2: sugar metabolism, epithelial transport, and barrier function, which likely reflect downstream events following receptor-ligand interaction. GWAS analysis revealed potential genetic contributions to GLP-2 half-life and receptor binding. Enteroendocrine cell loss was not identified in stunted Zambian children as has been observed for goblet and Paneth cells. Transcriptomic analysis suggests that GLP-2 has pleiotrophic actions on the intestinal mucosa in malnutrition, but further work is needed to dissect pathways leading to perturbations in nutrient sensing.

Reduced Fractional Absorption of Zinc in Children With Environmental Enteropathy in Zambia.

OBJECTIVES: We measured fractional absorption of zinc (FAZ) in children with environmental enteropathy (EE) and carried out transcriptomic analysis of biopsies from these children in order to compare FAZ to histology of intestinal biopsies, expression of zinc transporter genes, and biomarkers of enteropathy. METHODS: Fractional absorption of a standardized aqueous dose of zinc was measured by a dual isotope ratio technique in a cohort of children ages between 9 and 24 months in Lusaka, Zambia, who all had non-responsive stunting. Gene expression analysis was carried out on biopsies through RNA sequencing using an Illumina HiSeq2000 platform. RESULTS: All 33 children had histological features of environmental enteropathy and plasma zinc concentrations below the lower limit of normal. Measured FAZ ranged from 0.18 to 0.93; all values >0.55 were observed in girls. FAZ was negatively correlated with faecal myeloperoxidase (MPO) (ρ = -0.51, n = 17; P = 0.04) and faecal calprotectin (ρ = -0.50, n = 16; P = 0.05), but not blood biomarkers. Of 41 genes with known roles in zinc metabolism, only three metallothionein genes were significantly correlated with FAZ. CONCLUSIONS: Zinc homeostasis is impaired in children with environmental enteropathy, and was inversely correlated with mucosal inflammation. Reduced FAZ without specific changes in expression of most zinc transporter genes could be explained by reduced absorptive surface area due to villus/microvillus atrophy.