Gray platelet syndrome (GPS) is a rare autosomal recessive bleeding disorder characterized by thrombocytopenia, large platelets, and deficient alpha granules in platelets and megakaryocytes. The genetic cause of GPS was recently elucidated by our lab and others, and deleterious mutations were found in the gene NBEAL2 in several affected individuals. NBEAL2 stands for Neurobeachin-like 2 and is a large (30kb, 54 exon) gene on human chromosome 3p21.31. NBEAL2 is a member of a family of proteins that contain a BEACH (Beige and Chediak Higashi) domain which is a highly conserved domain that has been associated with protein functions such as vesicular trafficking, membrane dynamics, and receptor signaling. Very little is known about NBEAL2 other than its involvement with GPS. The relative expression of the NBEAL2 transcript, both in tissues and in subcellular compartments, is not known. It has previously been published that NBEAL2 protein coding isoforms are present in a variety of human tissues, but the abundance of NBEAL2 transcript in each tissue is not known. Furthermore, there is currently no available antibody that recognizes the 302 kDa NBEAL2 protein to validate that mRNA presence translates to protein expression. We have examined the relative transcript abundance of NBEAL2 in a human cDNA library and have validated a novel NBEAL2 antibody in a human megakaryocytic cell line (Dami) and human platelets. We obtained a human mRNA tissue library from Invitrogen which was reverse transcribed to cDNA. Tissues analyzed include bladder, brain, cervix, colon, esophagus, heart, kidney, liver, lung, ovary, placenta, prostate, skeletal muscle, small intestine, spleen, testes, thymus, thyroid, trachea, bone marrow, peripheral leukocytes, CD33+, and CD36+. To investigate relative transcript abundance, we used a Taqman qPCR probe designed to identify all six protein coding isoforms, as determined by Ensembl (http://uswest.ensembl.org). Highest NBEAL2 expression was seen in CD33+ cells, which was 54.3 fold higher than the tissue with lowest expression (skeletal muscle). Relatively high expression was also seen in the peripheral leukocytes, bone marrow, lung, esophagus, and cervix. Alternatively, NBEAL2 expression was low in the brain, despite the homology to the Neurobeachin (NBEA) brain specific protein. While high transcript abundance may infer function, relative protein expression is necessary to validate these findings. Therefore, we designed a novel NBEAL2 antibody against a 14 amino acid peptide (SLEPRRPEEAGAEVC) encoded by exon 1 of NBEAL2 that is 100% conserved between human and mouse. Western blot characterization of this antibody showed the expected approximately 300 kDa band in both soluble and insoluble Dami lysates and human platelet rich plasma lysates. Furthermore, the NBEAL2 antibody was also used for immunofluorescence of Dami cells to determine the approximate subcellular localization of the protein. Preliminary results suggest that NBEAL2 is localized to the cytoplasm, a finding that is consistent with a subcellular localization prediction program (Euk-mPLoc 2.0). In conclusion, we have determined the relative abundance of NBEAL2 transcript in several human tissues, and have begun to characterize a novel antibody against NBEAL2 using the human megakaryocytic Dami cell line and human platelets. Ongoing studies with this novel tool in the Nbeal2 knockout mouse model will likely provide new information about this elusive protein.


Di Paola:CSL Behring: Consultancy; Pfizer: DSMB, DSMB Other.

Author notes


Asterisk with author names denotes non-ASH members.