Molecular and Functional Characterization of Two Novel Missense Mutations in Iron Transport Protein Causing Hypochromic Microcytic Anemia and Hemosiderosis

Abstract 3202

Atransferrinemia is a rare autosomal recessive disorder characterized by hypochromic microcytic anemia and iron overload. It is extremely rare with 10 cases reported worldwide and only half of them have been characterized at the genetic level. In this report we describe two novel mutations in the transferrin gene found in two patients in two unrelated families from India.

The proband-I was a 9 year old girl who had severe hypochromic microcytic anemia requiring intermittent transfusions since the age of four years. She presented at 7 years of age with symptomatic anemia requiring blood transfusion not responding to oral iron therapy. She had pallor and mild hepato splenomegaly and her hemoglobin was 6.0g/dL with a MCV of 67.1fL. Her hemoglobin F (HbF) and hemoglobin A₂ (HbA₂) was normal and screening of beta globin gene did not reveal any mutation. Bone marrow examination done to rule out CDA & MDS showed a cellular marrow with absent iron stores. She didn't respond to oral iron supplements and her serum iron was 12ug/dL and TIBC was 110ug/dL with a ferritin of 3205ng/ml. Atransferrinemia was suspected because of a low TIBC and transferrin level of 5.7mg/dL confirmed the diagnosis. The proband-II was a 4 year old boy who had presented with hypochromic microcytic anemia and elevated ferritin. He was transfusion dependent from the age of two. His Hb was 6.4g/dl and MCV of 54.8fL with normal hemoglobin F (HbF) and hemoglobin A₂ (HbA₂) levels. His serum iron was 16ug/dL and TIBC 19 ug/dL with elevated ferritin (12910ng/ml). Low TIBC is correlated with very low transferrin level (TF-7.5mg/dl) found in this patient. DNA was extracted from the patients and parents by standard protocol. Both coding and adjacent regions of the exons in the transferrin gene (TF) were sequenced. Mutation analysis revealed the presence of a mutation c.1940 A>T in homozygous state resulting in a missense mutation p.Asp647Val in the C-lobe in proband-I. Both parents were heterozygous for this mutation and this change was absent in fifty healthy controls. Proband–II showed a homozygous c.1825 C>T variation in the transferrin gene resulting in p.Arg609Trp. Both parents were heterozygous for this mutation. These amino acids were highly conserved across species. When the protein sequence of TF was interrogated using SIFT and Polyphen algorithm for p.Asp647Val and p.Arg609Trp, both mutations were predicted to be pathological and deleterious. SPDBV modeling showed the p.Asp647Val will result in loss of interaction with a crucial amino acid Arg at 632 position which is essential for solvent accessibility and conformational change during iron release. There was a conformational change observed for the substitution of Arg by Trp at 609 position in TF protein by SPDBV modeling that may lead to instability or defective secretion and this has to be evaluated by invitro functional assays. To assess whether p.Asp647Val mutation leads to TF deficiency, in vitro TF production was assayed. T cells from the patient and controls were stimulated with phytohaemagglutinin and IL2 and the transferrin released was measured by ELISA. The amount of transferrin released from proband-I's T cells at 72hrs was 4.75ng/ml which was much lower when compared to transferrin secreted (72.14ng/ml) from control T cells. In vitro experimental observations clearly suggest that p.Asp647Val mutation in transferrin gene results in protein deficiency. As predicted, replacement of Aspartic acid with valine in C-lobe of transferrin leads to ineffective protein secretion. Proband-I was started on monthly plasma infusions. Her hemoglobin levels improved and she became transfusion independent. Oral iron chelation was started to reduce tissue iron overload. Proband-II has just been infused with plasma and the outcome is awaited. This is the first report from India where prevalence of thalassemia is high and identification of two cases underlines the importance of screening of atransferrinemia in cases with hypochromic microcytic anemia and iron overload in order to start appropriate treatment to reduce iron overload related complications and anemia.