No Mutations in the RPL11 Gene Detected in Greek Patients with Diamond Blackfan Anemia

Diamond Blackfan Anemia (DBA) is a rare congenital, bone marrow failure syndrome characterized by normochromic macrocytic anemia, reticulocytopenia and absence or insufficiency of erythroid precursors in normocellular bone marrow. Thirty to 40 percent of patients display also other somatic malformations. DBA affects 5–7 subjects per million live births and in ∼ 90% of the cases the diagnosis is made in infancy or early childhood. Thus, in Greece with a population number of around 10×10⁶ inhabitants, around 30–40 patients are expected. The disease is most commonly sporadic, but it can also be inherited as an autosomal dominant trait with incomplete penetrance. Heterozygous mutations in 9 ribosomal proteins genes have been reported in 50% of patients. Epidemiological studies in different populations showed that the most commonly affected genes are the RPS19 (with a reported frequency of 11–28%), the RPL5 (with a frequency of 9–21.4%) and the RPL11 (with a frequency of 4–9.3%).

The primary aim of this analysis is to assess the incidence of mutations in the RPL11 gene in Greek patients with DBA. τhis analysis is to complement the evaluation of the clinical and hematologic phenotype, as well as, the incidence of mutations in the RPS19 and RPL5 genes in these patients. Furthermore, the diagnostic value of Enzymatic Cleavage Mismatch Analysis (ECMA) in respect to the study of DBA was, also, assessed.

A questionnaire requesting data on clinical and hematological phenotype was sent to all the pediatric hematology, hematology and transfusion units in Greece. Informed consent was obtained from all patients and/or their family members participating in this study and genomic DNA was isolated from peripheral blood lymphocytes. PCR primers were specifically designed to amplify the whole coding region and the flanking intron/exon junctions of RPS19, RPL5 and RPL11 genes. RPS19, which bares the highest rate of mutations, was studied by both ECMA and direct sequencing. RPL5 and RPL11 genes were initially studied by ECMA. Direct sequencing was performed only in the samples that ECMA indicated the existence of a possible mutation.

Seventeen Greek patients, comprising 7 females and 10 males, with a mean age of 11.4±11 years, were referred for DBA molecular analysis. Congenital anomalies in different organs, including craniofacial, upper extremities, hands, eyes, heart and kidneys were observed in 66.7% of the patients. In regard to the clinical course, 4 patients were receiving chronic steroid therapy and 8 were on regular transfusions. One of the 8 transfusion-dependent patient developed thyroid cancer at the age of 46 years. One patient died from complications following bone marrow transplantation from an HLA-matched unrelated donor. Spontaneous resolution of the anemia was observed in one patient, one year after she had been referred, making the diagnosis of DBA on this patient questionable. The three remaining patients were lost to follow up.

As previously shown (Delaporta P et al.; Haematologica, 2011; 96(s2):345a), 6 patients (35.2%) were found to carry mutations on either the RPS19 gene (3 patients,) or the RPL5 gene (3 patients). Mutations c.C390G (p.'130X) and c.197_198insA (p.Y66X) detected in the RPL5 gene were novel. All the mutations in the RPS19 gene were identified by both ECMA and direct sequencing. Further molecular analysis on the RPL11 gene did not disclose any mutations on the six exons of the gene. Nevertheless, the molecular analysis revealed the previously described c.507+39 A>G variant in intron 5 (2 patients) and a novel c.264+49 A>G variant in intron 3 (1 patient).

Based on the reported frequency of RPL11 gene in previous population studies around the world, we would expect to identify 1–2 positive samples. No mutations at the RPL11 gene were revealed in Greek patients with DBA, but this may be due to the small sample size. Higher frequency of physical malformations and rates of transfusion dependency was observed compared to the ones reported from other countries. Finally, the consistency of the results between ECMA and direct sequencing support the high diagnostic value of the ECMA method in evaluating patients with DBA.

No relevant conflicts of interest to declare.