Large Deletions and Novel Mutations in Genes Coding for Ribosomal Proteins (RPL11, RPL35A, RPS19) in the Czech National Registry of Diamond-Blackfan Anemia (DBA)

Background: 50-70% cases of Diamond-Blackfan anemia (DBA) are underlied by mutations in genes coding for ribosomal proteins (RP), however, the etiology remains unexplained in the remaining patients. Recently, rare aberrations involving extraribosomal genes (e.g., GATA1, TSR2, MCM2, FLNB) have been reported as causative for DBA. Up to 10% of all DBA patients harbor large deletions involving some of the RP genes, which were not previously identified by sequencing.

Patients and Methods: Czech National Registry, containing data of 56 patients with DBA from Czechia and Slovakia (both children and adults), has a high detection rate of RP gene mutations (currently 82%). Using exome sequencing (Agilent SureSelect v6 Kit, HiSeq 2500 sequencer, Illumina) and array comparative genomic hybridization (aCGH, SurePrint G3 CGH+SNP microarray, 4x180K, Agilent), we examined periferal blood samples of patients without any proven mutation in RP genes. Identified mutations were verified by Sanger sequencing at the gDNA and cDNA level and their functional impact was studied in a cell culture (RPS11-deficient MRC-5 fibroblasts).

Results: Novel mutations, previously unpublished and unrecorded in gene databases, or large deletions involving RP genes were found in 5 patients with DBA:

1)In a 10-year-old female with growth and developlmental delay, severe skeletal anomalies (hypertelorism, high-arched palate, low-set ears, triphalangeal thumbs) and with a markedly elevated activity of erythrocytic adenosine-deaminase (eADA), a large deletion in 1p36 chromosomal region - arr[hg19] 1p36.12p36.11(22308520_24074348)x1 - involving the RPL11 gene was found. Depletion of RPL11 in a cellular model resulted in attenuation of proteosynthesis and cell cycle and in the disruption of nucleolar morphology.

2) In a 4-year-old female with transfusion-dependent DBA, large deletion of the 3q29 region - arr[hg38] 3q29(194309533_197837049)x1 - including the RPL35A gene was detected. The patient's height is at the 10th percentile, but her weight is below the 3rd percentile. Mild intellectual disability and both atrial and ventricular septal defect are present.

3) A 4,5-year-old male with transfusion dependency, iron overload and hepatopathy harbors mutation in RPS19 - hg38[19:41869031_C>T], RPS19 p.A58V. The patient's father with an identical mutation required repeated blood transfusions in childhood (until 6 years of age) due to hypoplastic erythropoiesis. To date, all parameters of his blood count are normal without any intervention, only his eADA level is slightly elevated.

All above mentioned patients are treated with leucine.

4) A 3-year-old male with normal phenotype harbors mutation hg38[19:41860841_A>T] in RPS19 leading to premature stop codon K23Stop.

5) Mutation in RPS19 : c.[3G>A], p.M1I in an 1 year old boy whose DBA is transfusion dependent and resistant to steroids.

Conclusion: Together with next generation sequencing, aCGH examination is essential for detection of novel genetic defects in DBA due to its capacity to detect larger chromosomal aberrations. Reports on 3q29 deletions involving RPL35A are scarce (only 7 cases published to date). Suggested connection between deletions, leading to haploinsufficiency of RP genes, and growth retardation (Kuramitsu et al., Blood 2012) was confirmed only for RPL11 but not for RPL35A in our study. Phenotypic differences between family members harboring the novel identical RPS19 aberration are probably influenced by epigenetic regulation and we have already observed this phenomenon in individuals with RPS7 and RPS19 mutation (Maceckova et al., ASH 2016).

Supported by: AZV 16-32105A, NPU LO 1304, GA15-13732S