Autosomal Recessive Diamond-Blackfan Anemia: Identification Of Mutations In MCM2 and Flnb

Diamond-Blackfan anemia (DBA) is a rare, congenital bone marrow failure syndrome characterized by severe anemia. DBA patients are generally diagnosed during infancy or early childhood and present with a high frequency of congenital anomalies, a predisposition to cancer and variable penetrance with severely affected and asymptomatic cases. Approximately 65% of DBA patients have heterozygous mutations or deletions in ribosomal protein (RP) genes encoding both large and small subunit proteins, resulting in autosomal dominant DBA. Current data suggest that the causal abnormalities in the remaining ∼35% of DBA patients are not in RP genes, leading to the hypothesis that mutations in non-RP genes may also cause DBA. Supporting this hypothesis, a recent study identified mutations in the gene encoding the transcription factor GATA1 in two DBA families (Sankaran VG et al. J Clin Invest. 2012 Jul 2;122(7):2439-43).

We used whole exome sequencing (WES) to identify candidate gene mutations in DBA patients who had normal RP gene sequences and no copy number variants by SNP array analysis. With informed consent, we performed WES on the proband, an unaffected sibling and their parents in four unrelated DBA families. For each individual in the study we obtained between 103 and 112 million sequence reads covering 91% of the coding exons with an average depth of coverage of 80X. Each family member had 8,000-10,000 variants that differed from the reference sequence. Filtering out the common variants from the 1,000 Genomes and ClinSeq databases reduced this number to ∼100 variants per individual. We then used the VarSifter program to evaluate the variants in the context of inheritance patterns. VarSifter analysis revealed no variants that were consistent with X-linked inheritance. We found 3-5 variants/proband that were consistent with either a sporadic autosomal dominant or with an autosomal recessive pattern of inheritance in two of the four families. Variants were prioritized for functional validation according to the Conserved Domain-based Prediction (CDPred) scores (used to predict the effect of amino acid substitution on the function of the protein), and for gene expression in erythroid cells. CDPred scores below -7 predict severe effects that may result in a truncated or non-functional protein. In one DBA family, we identified potential autosomal recessive mutations in the Mini Chromosome Maintenance Complex Component 2 (MCM2) (CDPred scores of -11) and Polymerase RNA III beta subunit (POLR3B) (CDPred scores of -10 and -30) as potential candidates. In another DBA family, we identified mutations in the Filamin B (FLNB) gene (CDPred scores of -9 and -7). RNA-Seq analysis of normal erythroid cells at defined stages of differentiation revealed that MCM2 and POLR3B mRNA levels are low in CD34+ progenitor cells, increase significantly in BFU-E, CFU-E, proerythroblasts, early and late basophilic erythroblasts, then decrease significantly in polychromatic and orthochromatic erythroblasts. FLNB mRNA is present at relatively high levels in CD34+ progenitor cells, BFU-E and CFU-E, and decreases significantly as erythrocyte maturation progresses.

We transduced CD34+ progenitor cells isolated from healthy donors with lentiviral vectors containing shRNAs targeting MCM2, POLR3B, or FLNB mRNA. RT-PCR analysis of RNA extracted from transduced cells demonstrated 60-90% knockdown of MCM2, POLR3B or FLNB mRNA. After 7 days of proliferation and 7 days of differentiation, we found that POLR3B knockdown cells showed no inhibition in the differentiation of CD41-/CD235+ erythroid cells compared to control, indicating POLR3B is an unlikely DBA candidate gene. In contrast, both MCM2 and FLNB depletion resulted in significant reductions in the number of CD41-/CD235+ erythroid cells, indicating these genes play important roles in erythropoiesis. Furthermore, when MCM2 shRNA transduced CD34+ progenitor cells were plated in semi-solid medium, CFU-GM colony numbers were normal, but BFU-E colony formation was significantly reduced, suggesting an erythroid-specific role for MCM2. In conclusion, we have identified mutations in the non-ribosomal protein genes MCM2 and FLNB in patients with DBA and demonstrated an important role for these gene products in erythropoiesis. These findings would represent the first autosomal recessive mutations identified in DBA patients.