SNX5, a Novel Gene Linked to Fanconi Anemia Causes Hematopoietic Failure When Knocked down in Zebrafish.

Sorting nexin 5 (SNX 5) is a member of the sorting nexin family, a diverse group of cellular trafficking proteins unified by the presence of a phospholipid binding domain (PHOX) which is involved in protein-protein interactions. While the function of SNX5 is unknown, there is evidence from yeast two hybrid studies that SNX5 binds to FANCA, and SNX5 is one of the 20 genes located on a region on mouse chromosome 2 associated with accelerated stem cell aging. We have recently shown that SNX5 is significantly higher expressed in CD34+CD33-CD38-Rho+ (Rho^hi) cells from umbilical cord blood (UCB) and bone marrow (BM), depleted of SCID repopulating cells (SRCs) than in CD34+CD33-CD38-cKIT+ Rho- cells (Rho^lo), containing all SRCs. A large scale high throughput functional analysis was carried out in zebrafish and genes differentially expressed between Rho^lo and Rho^hi cells were knocked down using morpholino (MO) antisense oligonucleotides. Morpholinos are a useful tool which can specifically inhibit the translation of target mRNA. 16 out of 70 genes knocked down, including snx5, demonstrated decreased circulating blood in the zebrafish injected. Further analysis has shown that MO knock down of snx5 in zebrafish (n=152) results in hematopoietic failure with normal vasculature, with normal expression of scl and gata-1 by in situ hybridization, indicating a defect at or beyond the hematopoietic stem cell stage. Quantitative RT-PCR was used to further confirm the phenotype. Levels of hemoglobin, l-plastin and myeloperoxidase mRNA in snx5 morphants compared to uninjected controls, were significantly lower, consistent with a multi-lineage hematopoietic differentiation defect. The decrease in circulating blood could be partially rescued by overexpressing human SNX5 cDNA, confirming specificity of the morphant phenotype. To determine whether SNX5 might be linked to Fanconi anemia (FA), we measured levels of SNX5 mRNA and protein in EBV transformed cell lines from patients with Fanconi-complementation group A (FANCA), -complementation group C (FANCC) or unknown complementation group (FANC-NX). We have found no significant difference between SNX5 mRNA expression in 11 cell lines compared to Jurkat controls. However, levels of SNX5 protein appeared lower in FANCA cells suggesting a possible role of SNX5 in FANCA, which is being evaluated further. Ongoing studies evaluating the role of SNX5 in normal human hematopoiesis will also be presented.