Assessment of SBDS Expression Patterns in Human Marrow Hematopoietic and Stromal Cells by Immunohistochemistry and Its Use as a Diagnostic Screen for Schwachman-Diamond Syndrome

Objective. Shwachman Diamond Syndrome (SDS) is an autosomal recessive syndrome characterized clinically by exocrine pancreatic insufficiency, bone marrow dysfunction, skeletal abnormalities and a predisposition to leukemia. Molecularly, it is characterized by mutations in the Shwachman-Bodian-Diamond-Syndrome (SBDS) gene encoded on chromosome 7. The majority of mutant alleles (74%) associated with SDS are the result of gene conversion events with an adjacent SBDS pseudogene, leading to truncation, splice site, and frameshift mutations and a marked decrease in SBDS expression. The remaining pathogenic alleles include frameshift and, rarely, missense changes¹. In this study, we examined the SBDS expression pattern in bone marrows from patients with SDS compared to normal controls or patients with other bone marrow failure syndromes using a new immunohistochemical (IHC) assay.

Methods. Archived bone marrow biopsy tissue sections from patients with SDS (21 specimens from 9 patients), idiopathic aplastic anemia (AA, 12 patients), Fanconi Anemia (FA, 4 patients), Diamond-Blackfan Anemia (DBA, 9 patients), Severe Congenital Neutropenia (SCN, 3 patients) and 13 normal controls were stained with an antibody directed against the C-terminus of SBDS. Four cell lineages (myeloid precursors, megakaryocytes, plasma cells and osteoblasts) were scored in a blinded fashion by two pathologists on a scale from 0 (negative) to 3 (strong). The scores from each cell lineage within each diagnostic group were averaged to yield an overall IHC score.

Results. We first characterized the SDS expression pattern in normal controls. Promyelocytes, early myelocytes, megakaryocytes, plasma cells, and osteoblasts all showed high levels of SBDS protein. In the myeloid lineage, SBDS expression decreased to undetectable levels in mature granulocytes, consistent with prior reports that SBDS is not necessary for terminal neutrophil differentiation. SBDS staining was not detectable in erythroid precursors or lymphocytes. We next compared SBDS IHC staining in these normal controls to that seen in SDS patients and patients with other bone marrow failure syndromes. The mean overall IHC score for SDS patients was 0.6 whereas it was 2.5 in normal controls, 2.23 in AA, 2.4 in DBA, 2.67 in FA and 2.38 in SCN. Four samples from two SDS patients with rare missense mutations in SBDS had moderate staining, consistent with previous studies that showed intermediate levels of SBDS expression in patients with missense mutations. When the analysis was restricted to patients harboring biallelic gene conversion SBDS mutations, the mean SBDS IHC score dropped to 0.14.

Conclusion. SBDS protein is highly expressed in early myeloid progenitors, megakaryocytes, and osteoblasts. This novel SBDS IHC assay may provide a rapid screen for the most common SBDS mutations.