RPS29 is Mutated in a Multi-Case Diamond Blackfan Anemia Family

Abstract ⁵¹¹

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome (IBMFS) characterized by red blood cell aplasia, variable physical anomalies, and increased risk of leukemia, myelodysplastic syndrome, lymphoma, and certain solid tumors, including osteosarcoma. DBA has been considered to be a disorder of ribosomal biogenesis because approximately 50% of cases are due to a mutation or deletion in 1 of 9 ribosomal protein genes (RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, RPS7, RPS10, or RPS26). However, mutations in GATA1, a hematopoietic transcription factor, have recently been reported to also cause DBA. DBA is inherited in an autosomal dominant manner, but de novo germline mutations have also been reported.

Our IBMFS cohort study conducts detailed clinical evaluations and medical record review of patients with DBA and their family members. We evaluated a large family with DBA in which mutation testing for the 10 known genes was negative. The male proband had steroid-responsive anemia as a child; he was in remission until he was treated with chemotherapy for squamous cell lung cancer at age 55 years. His healthy sister is an obligate carrier because her daughter was diagnosed with DBA as a child; her daughter had steroid-responsive anemia. The proband's maternal 1^st cousin had steroid-responsive DBA as a child which relapsed during pregnancy. Her 3 children (the proband's 2^nd cousins) had DBA. One died due to complications of transfusion-related iron overload, one had successful hematopoietic stem cell transplantation for steroid-refractory and transfusion-dependent anemia at age 26 years, and one has been off treatment and in remission for over 20 years. Except for the proband with lung cancer and a transfusion-dependent individual, all affected individuals had elevated red blood cell adenosine deaminase consistent with DBA.

We performed whole-exome sequencing on the 5 clinically affected individuals (a male proband, his affected first cousin, 2 affected second cousins, and an affected niece), his obligate carrier sister, and the unaffected father of the proband's niece. Genomic DNA was used to create an enriched multiplexed sequencing library (Nimblegen v2); this was followed by paired-end sequencing using an Illumina HiSeq™. We detected a total of 229,024 exonic nucleotide variants across this family, including 2,484 uncommon (minor allele frequency <5%) non-synonymous amino-acid substitutions. After removing variants that were present in publically available databases (1000Genomes, ESP, Kaviar, and dbSNP) and applying quality control filters, 731 candidate variants remained. We then evaluated these variants for autosomal dominant inheritance in this family. Remarkably, there was only 1 nonsynonymous variant present in all 5 affected individuals and in the obligate carrier, but absent from the unaffected non-obligate carrier parent. This nonsynonymous variant was present in exon 2 of the ribosomal protein S29 (RPS29) gene (chromosome 14q). This region of the RPS29 protein is very highly evolutionarily conserved. Bioinformatic analyses suggested that this variant is highly likely to affect protein function. SIFT, Polyphen 2, and Condel algorithms all predict this variant to be deleterious. RPS29 gene encodes the 40S ribosomal protein S29, which is a component of the small 40S ribosomal subunit and is important for ribosomal RNA processing and ribosome biogenesis. In addition, Zebrafish models of RPS29 mutations have significant defects in hematopoietic stem cell and red blood cell development. We are currently sequencing RPS29 in additional patients with DBA. In summary, whole-exome sequencing has allowed us to uncover another human DBA gene, RPS29, which will likely lead to improved understanding of this complex disorder.