A patient with a homozygous premature stop codon in PIK3R1 showed an early developmental block in B cell development but minimal effects in other organ systems.

IntroductionApproximately 85% of patients with early onset of infections, panhypogammaglobulinemia, and less than 2% CD19 + B cells in the peripheral circulation have X-linked agammaglobulinemia (1). This disorder is caused by mutations in Bruton tyrosine kinase (BTK) (2), a cytoplasmic tyrosine kinase that is activated by crosslinking of the pre-B cell and B cell antigen receptors (BCRs) (3). An additional 5%-7% of patients have rare autosomal recessive defects in components of the pre-BCR or BCR or in the downstream scaffold molecule B cell linker protein (BLNK) (4, 5). These genetic disorders all result in a block in B cell differentiation at the pro-B cell to pre-B cell transition, the stage at which the pre-B cell receptor is first expressed.We have recently described a group of 4 unrelated patients, 2 males and 2 females, with agammaglobulinemia and a very small number of B cells characterized by the lack of a BCR, but increased expression of CD19 (6). Bone marrow studies from these patients demonstrated a profound reduction in the number of CD19 + cells and a block in B cell differentiation at the common lymphoid precursor to pro-B cell stage of differentiation, a stage earlier than that seen in patients with BTK deficiency or mutations in components of the BCR signaling pathway.None of the 4 patients had a family history of immunodeficiency ( Figure 1A) or belonged to isolated populations or consanguineous families; therefore, we hypothesized that these

BackgroundAlthough advances in sequencing technologies have popularized the use of microRNA (miRNA) sequencing (miRNA-seq) for the quantification of miRNA expression, questions remain concerning the optimal methodologies for analysis and utilization of the data. The construction of a miRNA sequencing library selects RNA by length rather than type. However, as we have previously described, miRNAs represent only a subset of the species obtained by size selection. Consequently, the libraries obtained for miRNA sequencing also contain a variety of additional species of small RNAs. This study looks at the prevalence of these other species obtained from bone marrow aspirate specimens and explores the predictive value of these small RNAs in the determination of response to therapy in myelodysplastic syndromes (MDS).MethodsPaired pre and post treatment bone marrow aspirate specimens were obtained from patients with MDS who were treated with either azacytidine or decitabine (24 pre-treatment specimens, 23 post-treatment specimens) with 22 additional non-MDS control specimens. Total RNA was extracted from these specimens and submitted for next generation sequencing after an additional size exclusion step to enrich for small RNAs. The species of small RNAs were enumerated, single nucleotide variants (SNVs) identified, and finally the differential expression of tRNA-derived species (tDRs) in the specimens correlated with diseasestatus and response to therapy.ResultsUsing miRNA sequencing data generated from bone marrow aspirate samples of patients with known MDS (N = 47) and controls (N = 23), we demonstrated that transfer RNA (tRNA) fragments (specifically tRNA halves, tRHs) are one of the most common species of small RNA isolated from size selection. Using tRNA expression values extracted from miRNA sequencing data, we identified six tRNA fragments that are differentially expressed between MDS and normal samples. Using the elastic net method, we identified four tRNAs-derived small RNAs (tDRs) that together can explain 67 % of the variation in treatment response for MDS patients. Similar analysis of specifically mitochondrial tDRs (mt-tDRs) identified 13 mt-tDRs which distinguished disease status in the samples and a single mt-tDR which predited response. Finally, 14 SNVs within the tDRs were found in at least 20 % of the MDS samples and were not observed in any of the control specimens.DiscussionThis study highlights the prevalence of tDRs in RNA-seq studies focused on small RNAs. The potential etiologies of these species, both technical and biologic, are discussed as well as important challenges in the interpretation of tDR data.ConclusionsOur analysis results suggest that tRNA fragments can be accurately detected through miRNA sequencing data and that the expression of these species may be useful in the diagnosis of MDS and the prediction of response to therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1929-y) contains supplementary material, which is available to authori...

Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders of the elderly that carry an increased risk of progression to acute myeloid leukemia (AML). Since small non-coding RNAs (sRNAs), including microRNA (miRNAs), act as regulators of cellular differentiation, we hypothesized that changes to sRNAs might be implicated in the progression of MDS to AML. We conducted sRNA sequencing on three sets of patients: Group A (MDS patients who never progressed to AML); Group B (MDS patients who later progressed to an AML); and Group C (AML patients with myelodysplasia-related changes, including patients with a known preceding diagnosis of MDS). We identified five miRNAs that differentiated Groups A and B, independent of bone marrow blast percentage, including three members of the miR-181 family, as well as differential patterns of miRNA isoforms (isomiRs) and tDRs. Thus, we have identified sRNA biomarkers that predict MDS cases that are likely to progress to AML.

Expression of a BCR is critical for B-cell development and survival. We have identified 4 patients with agammaglobulinemia and markedly reduced but detectable B cells in the peripheral circulation. These B cells have an unusual phenotype characterized by increased expression of CD19 but no BCR. The cells are positive for CD20, CD22, and CD38, but not for Annexin 5 or activation markers, including CD69, CD83, or CD86. EBV lines derived from these B cells lack functionally rearranged immunoglobulin heavy-chain transcripts, as shown by PCR-rapid amplification of cDNA ends (PCR-RACE). Analysis of BM from 2 of the patients showed a severe reduction in the number of pro-B cells as well as pre-B cells. Functionally rearranged heavy-chain transcripts were identified, indicating that machinery to rearrange immunoglobulin genes was intact. Flow cytometry of B-lineage cells suggested accelerated acquisition of maturation markers in early B-cell precursors and increased phosphorylation of signal transduction molecules. Further, expression of TdT, a molecule that is normally down-regulated by a functional pre-BCR complex, was decreased. We hypothesize that the accelerated maturation, increased expression of CD19, and lack of a BCR were due to the constitutive activation of the BCR signal transduction pathway in these patients. (Blood. 2011;118(7):1828-1837) Introduction B-cell development is critically dependent on appropriate signaling through the pre-BCRs and BCRs. Mutations that decrease the function of any of the components of these receptors, including heavy chain, Ig␣, Ig␤, and the proteins that make up the surrogate light chain result in a severe block in B cell differentiation at the pro-B to pre-B-cell transition. [1][2][3][4][5] Defects in proteins required for downstream signaling through these receptors, including Btk and BLNK, also result in a selective B-cell deficiency. [6][7][8] However, mutations in these proteins do not account for all of the patients with isolated defects in B-cell development.Premature or excessive signaling through the pre-BCR or BCR may also cause a reduction in the number of peripheral B cells. Early studies in transgenic mice showed that expression of the membrane form of a rearranged immunoglobulin heavy chain resulted in markedly reduced numbers of B-lineage cells in the BM and in the periphery. 9 This was attributed, at least in part, to rapid transit through early stages of differentiation. Rearranged heavy chains encoding high-affinity or activating antibodies for auto-antigens result in clonal deletion of immature B cells. 10 Enhanced activation of the BCR pathway also causes a reduction in the number of B cells. Mice bearing a transgene for a constitutively active form of Btk (E41K) have a B-cell deficiency that is more severe than that seen in mice that lack Btk. 11 More indirectly, increased expression of CD19, a molecule that enhances signaling through the BCR, also results in a marked decrease in the number of peripheral B cells. 12 Genetic alterations that enhance B-cell s...