Bone marrow immunophenotyping by flow cytometry in refractory cytopenia of childhood.

Refractory cytopenia of childhood is the most common type of childhood myelodysplastic syndrome. Because the majority of children with refractory cytopenia have a normal karyotype and a hypocellular bone marrow, differentiating refractory cytopenia from the immune-mediated bone marrow failure syndrome (very) severe aplastic anemia can be challenging. Flow cytometric immunophenotyping of bone marrow has been shown to be a valuable diagnostic tool in differentiating myelodysplastic syndrome from non-clonal cytopenias in adults. Here, we performed the first comprehensive flow cytometric analysis of immature myeloid, lymphoid cells and erythroid cells, and granulocytes, monocytes, and lymphoid cells in bone marrow obtained from a large prospective cohort of 81 children with refractory cytopenia. Children with refractory cyotopenia had a strongly reduced myeloid compartment, but not as severe as children with aplastic anemia. Furthermore, the number of flow cytometric abnormalities was significantly higher in children with refractory cytopenia than in healthy controls and in children with aplastic anemia, but lower than in advanced myelodysplastic syndrome. We conclude that flow cytometric immunophenotyping could be a relevant addition to histopathology in the diagnosis of refractory cytopenia of childhood. (The multi-center studies EWOG-MDS RC06 and EWOG-MDS 2006 are registered at clinicaltrials.gov identifiers 00499070 and 00662090, respectively).

Human telomere disease due to disruption of the CCAAT box of the TERC promoter.

Mutations in the coding region of telomerase complex genes can result in accelerated telomere attrition and human disease. Manifestations of telomere disease include the bone marrow failure syndromes dyskeratosis congenita and aplastic anemia, acute myeloid leukemia, liver cirrhosis, and pulmonary fibrosis. Here, we describe a mutation in the CCAAT box (GCAAT) of the TERC gene promoter in a family in which multiple members had typical features of telomeropathy. The genetic alteration in this critical regulatory sequence resulted in reduced reporter gene activity and absent binding of transcription factor NF-Y, likely responsible for reduced TERC levels, decreased telomerase activity, and short telomeres. This is the first description of a pathogenic mutation in the highly conserved CCAAT box and the first instance of a mutation in the promoter region of TERC producing a telomeropathy. We propose that current mutation-screening strategies should include gene promoter regions for the diagnosis of telomere diseases. This clinical trial was registered at www.clinicaltrials.gov as #NCT00071045.

Extreme elevation of serum growth hormone-binding protein concentrations resulting from a novel heterozygous splice site mutation of the growth hormone receptor gene.

BACKGROUND/AIMS: Circulating growth hormone-binding protein (GHBP), in humans, is the proteolytic product of the growth hormone receptor (GHR). We investigated a prepubertal male subject who was of short stature, but who had a markedly elevated serum level of GHBP. METHODS: Serum and DNA from the patient and his mother were analyzed. RESULTS: Both the patient and mother had serum GHBP concentrations over 100-fold higher than normal, by assays, and Western and ligand blot analysis. Sequencing of the GHR gene revealed a novel heterozygous C>A transversion at position 785-3 in the acceptor splice site of intron 7. CONCLUSION: In silico analysis of the altered sequence suggested that 785-3(C>A) is a splicing mutation, with either retention of intron 7 or the skipping of exon 8. The consequence is a truncated GHR lacking the transmembrane domain (encoded by exon 8) and the cytoplasmic domain. We hypothesize that this GHR variant cannot anchor to the cell membrane, and the continual secretion into the circulation explains the elevated levels of serum GHBP detected in the patient and his mother. Despite this mutation, the presence of the wild-type GHR allele, presumably, permits some normality in GH-induced action.