Abstract 2369

Diamond-Blackfan anaemia (DBA) is a rare autosomal dominant disorder associated with inactivating mutations in ribosomal protein (RP) genes, causing defects in erythroid progenitor and precursor cell development. Many cases are due to de novo mutations and in family cases there is often clinical heterogeneity due to variable penetrance. Mutations in RPS19 account for 25% of all DBA cases and single nucleotide variations (SNV), indels and allele-loss deletions have been found in 11 other RP genes in a further ∼50% of patients. Around 25% of patients with DBA have no identifiable mutations. Given that (with the exception of 2 cases with GATA1 mutations) all mutations in DBA characterised so far affect RP genes, it is likely that mutations in one of the 80 RP genes will be eventually identified in a significant proportion of the patients. Current screening methods are primarily based on Sanger sequencing on a per-exon/per-gene basis, with the associated time, labour and cost restrictions. We therefore aimed to evaluate high-throughput sequencing technology, including a bespoke target enrichment platform, to screen all 80 known RP genes to facilitate rapid, cost-effective identification of DBA associated mutations. DNA was extracted from peripheral blood samples that had been referred to Imperial Molecular Pathology for DBA screening from 10 individuals, including 3 family pairs: affected mother and daughter; 2 affected siblings; and another sibling pair, one of whom was unaffected/low-penetrance (no defining clinical symptoms, except for high adenine deaminase). Only one patient had a known mutation (RPS19 c.280C>T) and was included as a control. Agilent SureSelect XP was used for the target enrichment, which employed a custom designed tiled-RNA bait hybridisation solution to capture the target genes, including non-masked intronic regions and 500bp of flanking sequence. The DNA was sheared using a Covaris e220, QC was performed via QIAxcel capillary electrophoresis and the hybridisation was carried out at 65°C for 48h. Individual libraries were quantified using qPCR against the supplied standard curve and pooled proportionally. The sequencing was performed on an Illumina MiSeq, using 150bp paired-end reads and multiplexed using the supplied ScriptSeq barcodes. The sequencing reads were aligned to the build 37 reference genome using BWA software, and the variant calls made using GATK. Annovar was used for functional annotations of the variants. Protein truncating mutations were found in RP genes in 7 of the 10 samples, including the positive control and 6 of the 8 clinically confirmed DBA patient samples., All mutations were in RP genes previous described as being involved in DBA, although 3 affected novel codons: RPL5 c.G244T (stop-gain SNV; novel; mother-daughter pair); RPL5 c.166_169delACAA (frameshift); RPS10 c.C337T (stop-gain SNV); RPL11 c.472–473delAA (frameshift; novel); RPS26 c.212–213insA (frameshift; novel). Validation was by Sanger sequencing and further confirmation testing will include unaffected family members. The remaining 2 DBA patients, a brother-sister pair, showed no definable mutations in the captured regions and neither did the unaffected/low-penetrance sibling of the RPS10 patient. In summary, a rapid and cost effective methodology for screening genetic lesions associated with the causation of DBA is warranted, especially given the magnitude of attaining global coverage by conventional techniques. Whole-gene enrichment followed by multiplexed runs on a bench-top class high-throughput sequencing platform is arguably the approach of choice; although as the cost of exome and even genome sequencing continues to fall, these may well become realistic options in the coming few years. This work is ongoing, with a second group of 10 samples already sequenced and undergoing analysis, and bioinformatic refinements, especially for the detection of larger deletions, may yet yield results for the two undetected samples. These preliminary results suggest that high throughput sequencing technology with a bespoke target enrichment platform for RP genes is a feasible, efficient and relatively rapid diagnostic tool for detection of causative mutations DBA.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.