A Mild DBA Phenotype Associated with a Non-Canonical Splice Site Mutation in RPS26

A 37-years-old Italian female came to the observation of an adult hematology unit for the first time because of an undefined form of congenital anemia. The patient's unrelated parents and her sister were apparently healthy. Hypochromic anemia was observed for the first time at the age of 11 months and treated with one blood transfusion. Oral iron supplementation was started but was ineffective (Hb around to 7 g/dL). At that time, Fanconi's anemia was excluded by cytogenetic analysis, as also DBA The patient showed normal growth and somatopsychic development. Hematology showed moderate macrocytic anemia (MCV ranging from 115-121 fl, Hb levels ranging from 7 to 9 g/dL) with anisocytosis and hypochromia, inadequate reticulocytosis, normal bilirubin and haptoglobin, increased serum ferritin, increased HbF (7-8%), slightly increased erythropoietin, but proportional to anemia, and erythroblastic hyperplasia with variable dyserythropoiesis on bone marrow aspiration and bone marrow biopsy. From the age of 20 onwards, Hb levels ranged between 6.8 and 7.5 g/dL, without transfusion support and allowing a normal working activity. The patient underwent cycles of steroid treatment with moderate benefit. Abdomen ultrasound analysis showed mild steatosis with presence of gallstones, and no signs of focal lesions. At the age of 38 years a first spontaneous pregnancy resulted in intrauterine loss of a female fetus at 21 weeks of gestation because of umbilical cord strangulation. During pregnancy worsening of anemia (Hb 5.8 g/dL) required four blood transfusions. A second spontaneous pregnancy resulted in embryonic loss. Subsequently, she was subjected to several IVF attempts: at 47 years a pregnancy resulted in embryonic loss at 8 weeks of gestation, whereas at 49 years a further pregnancy resulted in a healthy male now 9 years old. Hb levels around 8g/dL were maintained by 20 transfusions, starting at 4 months of gestation. The patient is now 59 and has not developed any tumors, so far.

To ascertain the nature of anemia, the patient underwent several investigations: RBC morphology showed anysopoikilocytosis with 10% elliptocytes and 6% spherocytes. Osmotic fragility tests, Eosin-5' maleimide (EMA)-binding test, unstable hemoglobins, SDS-PAGE electrophoresis of red cell membrane proteins as well as the assay of RBC metabolism were normal, except for a decrease of pyruvate kinase/hexokinase (PK/HK) ratio (as sometime happens in presence of dyserythropoiesis).

Targeted-NGS sequencing using a panel of 48 genes associated with congenital hemolytic anemias, including RBC membrane defects, RBC metabolism and congenital dyserythropoietic anemias was performed without finding any pathogenic variant. Clinical Exome Sequencing analysis showed the presence of a heterozygous mutation in RPS26 gene: c.3+5G>C. Reverse transcription PCR on total RNA extracted from the patient's PBMC showed that the variant is associated with activation of a cryptic splice site. The aberrant transcript, which is not present in controls, lacks the last 18bp of exon 2 including the ATG (hg19). The fact that the RPS26: c. 3+5G>C mutation does not alter the canonical GT suggests that the mutation may be leaky. This would explain the patient's mild phenotype. A minigene assay is needed to ascertain this hypothesis.

In conclusion, if not identified in infancy, milder forms of DBA may be under- or misdiagnosed, further underlying the role of NGS in diagnosis of atypical inherited anemias. The present case further supports the hypothesis that mutations in RPS26 may be associated to a lower cancer risk in DBA.