Diamond-Blackfan Anemia Syndrome Research Articles

Case report: Rethinking NGS analysis in diagnosing Diamond-Blackfan anemia syndrome

Diamond-Blackfan anemia syndrome (DBAS) is a rare inherited bone marrow failure (BMF) syndrome characterized by erythroid aplasia, congenital malformations, and cancer predisposition. With its genetic heterogeneity, variable penetrance and expressivity, DBAS poses significant diagnostic challenges, necessitating advancements in genetic testing for improved accuracy. Here, we present the case of an 18-year-old male with a long-standing macrocytic anemia that remained undiagnosed despite standard whole exome sequencing (WES). Revisiting a family-trio WES analysis with clinical insight led to the identification of a likely pathogenic variant in the Ribosomal Protein S17 (RPS17) gene, previously masked due to analytical challenges and conservative filter settings. This variant, an initiation codon mutation, was confirmed in heterozygosity in both the proband and his mother through Sanger sequencing. Comprehensive imaging studies showed no malformations or organ anomalies in either individual, except for mild esophageal stenosis observed in both. RPS17 mutations, particularly those affecting the initiation codon, have previously been linked to the DBAS phenotype, but strong pathogenic association has not yet been firmly established. Our case warns of potential underdiagnosis of RPS17 variants in DBAS, highlighting the importance of clinical context and interdisciplinary collaboration in interpreting WES data to avoid false-negative results.

An atypical form of 60S ribosomal subunit in Diamond-Blackfan anemia linked to RPL17 variants.

Diamond-Blackfan anemia syndrome (DBA) is a ribosomopathy associated with loss-of-function variants in more than 20 ribosomal protein (RP) genes. Here, we report the genetic, functional, and biochemical dissection of 2 multigenerational pedigrees with variants in RPL17, a large ribosomal subunit protein-encoding gene. Affected individuals had clinical features and erythroid proliferation defects consistent with DBA. Further, RPL17/uL22 depletion resulted in anemia and micrognathia in zebrafish larvae, and in vivo complementation studies indicated that RPL17 variants were pathogenic. Lymphoblastoid cell lines (LCLs) derived from patients displayed a ribosomal RNA maturation defect reflecting haploinsufficiency of RPL17. The proteins encoded by RPL17 variants were not incorporated into ribosomes, but 10%-20% of 60S ribosomal subunits contained a short form of 5.8S rRNA (5.8SC), a species that is marginal in normal cells. These atypical 60S subunits were actively engaged in translation. Ribosome profiling showed changes of the translational profile, but those are similar to LCLs bearing RPS19 variants. These results link an additional RP gene to DBA. They show that ribosomes can be modified substantially by RPL17 haploinsufficiency but support the paradigm that translation alterations in DBA are primarily related to insufficient ribosome production rather than to changes in ribosome structure or composition.

Generation of iPSC lines and isogenic gene-corrected lines from two individuals with RPS19-mutated Diamond-Blackfan anemia syndrome

Diamond-Blackfan anemia syndrome (DBAS) is an inherited bone marrow failure disorder that typically presents in infancy as hypoplastic anemia and developmental abnormalities in approximately 50% of cases. DBAS is caused by haploinsufficiency in one of 24 ribosomal protein genes, with RPS19 mutations accounting for 25% of cases. We generated iPSC lines from two patients with different heterozygous RPS19 mutations (c.191T > C and c.184C > T) and isogenic lines in which the mutations were corrected by Cas9-mediated homology directed repair.

Whole Genome Sequencing of Diamond Blackfan Anemia Syndrome Patients Detects Mutations That Alter mRNA Splicing

Diamond Blackfan anemia syndrome (DBAS) is a rare, heritable bone marrow failure syndrome characterized by severe macrocytic anemia, congenital anomalies and predisposition to cancer, most often diagnosed during infancy. More than 98% of DBAS patients with a molecular diagnosis have mutations in a gene encoding one of the ~80 ribosomal proteins (RP) leading to haploinsufficiency. A molecular diagnosis in a patient with DBAS is critical for a definitive diagnosis, the identification of compatible related transplant donors, and developing reproductive strategies for families. Targeted sequencing of RP genes, single nucleotide polymorphism comparative genome hybridization (SNP array) to detect >30 kb deletions (Farrar et al. Blood. 2011) and exome sequencing (WES) (Ulrisch et al. Am J Hum Genet. 2018) has identified RP mutations in ~80% of patients, leaving ~20% of patients with DBAS without a molecular diagnosis.Targeted sequencing and WES focus on only coding sequences. We hypothesized that remaining 20% of DBAS mutations were in the non-coding regions of RP genes, such as promoters or introns. To test this hypothesis, we collected DNA with informed consent for whole genome sequencing (WGS) analysis from 14 patients with no molecular diagnosis after targeted sequencing, SNP array or WES. On average, we aligned ~3.2x10 7 paired end reads of 250 base pairs for each patient (~65X coverage). We focused our analysis on the sequences in and around the RP genes. To identify deletions, we used a suite of detection tools: DELLY, GRIDSS, MANTA, and LUMPY. More than 90% of deletions identified by any 2 of these tools were confirmed by PCR.We identified 5 deletions in the introns of RP genes, ranging from 11 to 467 base pairs in length, which we hypothesized disrupted splicing of the nascent RNA transcript. To test this, we created minigenes in which we replaced exon 2 of a gamma globin gene with either the WT or mutant RP exon. All wild type exons spliced normally. A 467 base pair deletion in RPL27 exon 3 was sufficient to prevent the correct splicing of that intron. Examination of the eCLIP data for RNA binding proteins revealed that spliceosome complex proteins (including SF3B1, SF3B4 and EFTUD2) and Dead-box RNA helicases bind in the deleted region. A 28 base pair deletion in exon 3 of RPL6 removes a polypyrimidine tract that is a critical part of the 3' splice junction consensus sequence, which we presume is also deleterious. The other 3 intronic deletions did not disrupt splicing.We also identified 2 causative point mutations. A point mutation 5 bases into intron 1 of the RPS26 gene changes a base in the 5' splice donor consensus sequence, which activated a cryptic splice donor in the 5' untranslated region. This aberrant splice removes the ATG initiation codon causing an untranslatable RNA. In another patient, we identified a mutation in exon 1 of the RPS27 gene, judged to be a benign amino acid change. This mutation disrupted splicing.by activating a cryptic splice donor site in the 5' untranslated region which removes the ATG initiation codon and causes a frame shift.We were referred two patients with possible duplications of the RPL35a gene. To identify duplications, we employed MinION long read single molecule sequencing. We had an average read length of ~ 6-10kb with the longest read being 1.3Mb. Overall coverage was >85X. We used minimap2 to align the reads to the reference human genome and used SNIFFLES to call the variants. One patient was the parent of DBAS-affected patient with no history of anemia. In this patient, we identified a duplication of 400 kb that included the entire RPL35a region along with genes on either side. We conclude that this duplication is not likely to cause DBA. The second patient was diagnosed with DBAS. In this patient, we identified a duplication of 4 kb including exons 1 and 2 of RPL35a We conclude that this duplication disrupts the RPL35a gene and is a likely cause of DBA.Whole genome sequencing of 15 DBAS patients identified 5 likely causative mutations in RP genes, confirming that most genetically undiagnosed cases of DBAS will involve known genes encoding RP. We conclude that the pipeline for obtaining a molecular diagnosis for DBAS from targeted sequencing, SNP array, and exome sequencing to whole genome sequencing. DisclosuresVlachos: Novartis: Membership on an entity's Board of Directors or advisory committees. Lipton: Celgene: Membership on an entity's Board of Directors or advisory committees.

Whole Genome Sequencing Identifies Small Deletions in Ribosomal Genes Causing Diamond Blackfan Anemia

Diamond Blackfan Anemia Syndrome (DBA) is a rare, congenital bone marrow failure syndrome characterized by severe macrocytic anemia, most often diagnosed during infancy. Congenital anomalies and predisposition to cancer are also important features of DBA. Establishment of a molecular diagnosis in a patient with DBA is critical to determine treatment strategies (i.e. the identification of compatible related transplant donors), as well as developing reproductive strategies for genetically at risk families. The overwhelming majority (>98.75%) of DBA patients with a molecular diagnosis have mutations in a Ribosomal Protein (RP) gene. Targeted and exome sequencing (WES) strategies can identify RP mutations in >70% of DBA patients (Ulrisch et al. Am J Hum Genet. 2018). Single Nucleotide Polymorphism Comparative Genome Hybridization (SNP array) detects >30 kb deletions of RP genes (which cannot be identified by sequencing) in ~10% of DBA patients (Farrar et al. Blood. 2011), leaving ~20% of DBA patients without a molecular diagnosis. We hypothesized that smaller copy number variants (CNVs - either insertions or deletions) in RP genes that are below the limit of detection of SNP array are responsible for the remaining 20%. To test this hypothesis we collected DNA with informed consent for whole genome sequencing (WGS) analysis from 6 patients who had no mutations detected by WES or SNP array. On average, we aligned ~1x1010paired end reads of 250 base pairs for each patient (~83X coverage of the genome). The aligned sequences were analyzed for CNVs using two independent software packages. Delly analyzes the two ends of each sequence read and maps them to the current human reference genome. Read ends that map further apart than expected are flagged as potential CNVs. CNVkit estimates regions of copy loss by changes in average sequencing depth. Using relatively relaxed thresholds in Delly and CNVkit we identified ~100 candidate CNVs in each patient. We filtered out CNVs present in public databases and focused on those CNVs in the region of the RP genes. This analysis identified 2-5 potential RP gene associated CNVs in each patient. We designed PCR primers that flanked each putative CNV and confirmed at least one RP CNV in all 6 patient DNAs. At this time, the CNVs in two patients are in the process of evaluation. We have validated causative RP CNVs in the other 4 patients, representing one known and three novel DBA genes. One patient had a 464 bp deletion in 3rdintron of the RPL27 gene, which is mutated in other DBA patients. We hypothesized that the deletion caused a splicing defect. Using a mini gene in which the second intron of the gamma globin gene was replaced with the 3rdintron of either the wild type or mutant RPL27 gene, we showed that the mutant exon was not spliced. An alternative hypothesis, that the deletion removed an enhancer element, was also tested, but no enhancer activity was detected. We conclude that the RPL27exon deletion causes aberrant splicing leading to an unstable RPL27 mRNA and haploinsufficiency of RPL27. A second patient had a 3.5 kb deletion at the 3' end of the RPS5 gene, including the stop codon and poly A addition site. We hypothesized that the lack of the 3' processing signals would lead to an unstable mRNA. To test this hypothesis we generated MYC and FLAG tagged wildtype and 3' deleted RPS5 genes and co-transfected them into 293T cells. Regardless of the tag used, RT-PCR analysis showed a severe reduction in the mutant mRNA levels. Western Blot analysis demonstrated that only the wild type protein was expressed, leading to the conclusion that the RPS5 truncation led to an unstable RPS5 mRNA and haploinsufficiency of RPS5. A third patient had a 28 kb deletion that removes the RPS9 gene. shRNA knockdown of RPS9 mRNA in normal CD34+ cells inhibited erythroid differentiation, leading to the conclusion that RPS9 deficiency causes DBA. Finally, we observed a 3 bp insertion in exon 6 of the RPL14 gene. The deletion adds an alanine residue to a string of 10 alanines in the wild type allele. We confirmed the insertion by targeted sequence analysis of patient DNA. Our data show that WGS can identify small CNVs that cause DBA in at least 2/3 of patients who do not have a mutation detectable by other methods. We believe that WGS analysis following targeted sequencing, SNP array and WES can identify virtually all DBA mutations. With declining WGS costs, we recommend adding WGS to the molecular diagnostic pipeline for genetic testing of DBA. Disclosures Farrar: Novartis: Research Funding. Vlachos:Novartis: Other: Steering committee member.