Low affinity hemoglobinopathy (<scp>H</scp>b <scp>V</scp>igo) due to a new mutation of beta globin gene (c200 <scp>A</scp>&gt;<scp>T</scp>; <scp>L</scp>ys&gt;<scp>I</scp>le). A cause of rare anemia misdiagnosis
Authors: María del Mar Mañú Pereira, Paloma Ropero, Carmen Loureiro, Joan‐Lluis Vives Corrons
Journal: American Journal of Hematology (2017)

A 12-year-old girl of Spanish origin was referred for chronic anemia since 11 months of age. Her complete blood count (CBC) showed a hemoglobin (Hb) concentration of 103 g/L to 106 g/L, with normal reticulocyte count (47-54 ×109/L). Other hematological and biochemical parameters, including ferritin (39 ng/mL) and transferrin saturation index (TSI: 31%) were normal, and only serum erythropoietin (Epo) concentration was decreased (8.0 mU/mL, RV: 9-19). All known causes of anemia for the age of the patient were investigated, including maturation factors, liver and renal concomitant disorders, prolactin, thyroid and growth hormones. Moreover, RBCs and bone marrow morphology, hemolytic tests, including RBC enzymes and membrane studies were also normal. Hb analysis by HPLC showed a structural abnormal Hb variant in heterozygous condition (HbX/HbA + HbA) with a relative quantification of 54.5%, indicating a beta-globin chain mutation (Figure 1). Abnormal HbX and Hb A were well separated at alkaline pH electrophoresis, since Hb X moved faster than Hb A (Figure 2A), but not at acid pH (Figure 2B). In order to investigate HbX mutation, whole blood samples and genetic material were obtained from the girl and her parents, that were clinically normal and with normal HPLC pattern. Sequencing of beta globin gene was also performed in the patient and her parents, demonstrating only in the patient, an heterozygous condition of a missense mutation, HBB c200 A > T responsible for the amino acid change Lys > Ile at codon 66 (Figure 3). Mutations at codon CD66 are responsible for low oxygen affinity and anemia due to concomitant Hb instability.1 In our patient, heat and isopropanol stability tests were normal, but the oxygen–hemoglobin affinity study (PR. Hospital Clínico San Carlos) showed a rightward shift of the oxyhemoglobin dissociation curve (ODC) with an increased P50 value of 32 mm Hg (RV: 26-29 mm Hg), characteristic of low affinity hemoglobinopathy. Due to patient's origin, this new, not previously described Hb variant, was named "Hb-Vigo," and since both parents showed a normal HBB gen genotype, a "de novo" origin of the mutation was hypothesized. Unfortunately, parenteral relationship could not be ruled out by a mosaicism study. Hemoglobin analysis by HPLC showing the abnormal peak responsible for 45.5% of beta gene production Alkaline (A) and acid (B) electrophoresis Nucleotide sequence of beta globin gene from the patient amplified by PCR and sequenced. Sequences of codons are shown, and heterozygosity at codon CD 66 AAA > ATA is indicated with an arrow. Mutations leading to abnormal structure or to a decrease in the alpha or beta globin chains levels will result in structural hemoglobinopathies (Hb variants) and thalassemia, respectively.1, 2 More than 1600 Hb variants and thalassemias are catalogued on the Globin Gene Server database (HbVar, http://globin.bx.psu.edu).3, 4 Functional and genetic analysis of these variants has generated valuable insights into structure–function relationships within the Hb molecule, with significant clinical consequences. Some very rare mutations in globin chains change the affinity of the Hb molecule for oxygen (O2), thereby disturbing the normal loading of O2 in the lungs and delivery of oxygen to the tissues.5 These mutations show an autosomal dominant pattern of inheritance, and are characterized by an increase or a decrease of Hb–O2 affinity, leading to high affinity hemoglobinopathies (HAH) or low affinity hemoglobinopathies (LAH), respectively. HAH become saturated at lower O2 pressures and stimulate erythropoietic drive by inhibiting O2 tissue delivery, thus resulting in erythrocytosis.6 On the contrary, LAH show low saturation index, inhibit erythropoietic drive by stimulating O2 tissue delivery, and result in a decrease of erythropoietin (Epo), RBC production, and a compensatory anemia. Accordingly, LAH variants should be considered in the differential diagnosis of patients with rare anemia and/or unexplained neonatal cyanosis.7 Moreover, in clinical practice, the diagnosis of LAH is very difficult to diagnose due to the low clinical expression of this rare disease, and the lack of specific diagnostic laboratory tests.8 According to the Globin Gene Server database,3 the present case is the first description of this specific nucleotide change, despite three different mutations have been described so far affecting the lysine at codon 66.9 Interestingly, from these three cases, two presented with slight chronic anemia, due to concomitant Hb instability, and one without clinical manifestations and found on occasion of an interference of HbA1c measurement. Our observation of reduced O2 affinity in Hb (beta 66(E10) Lys > Ile) provides further support to the functional importance of this beta 66 lysine residue for oxygen binding, as demonstrated by the findings of the other three Hb variants already described.3, 4 Our patient provides the first example of a beta globin chain mutation HBB c200 A > T (CD66 Lys > Ile), responsible for a LAH associated with chronic anemia not due to hemoglobin instability. Since after an exhaustive study, no known causes of anemia were found in the patient, it has assumed that the anemia is compensatory for the increased O2 delivery to the tissues and produced by a decrease of Epo synthesis. Compensatory anemia due to reduced erythropoietic drive was firstly described by Stamatoyannopoulos et al. in 1969,10 and, to our knowledge, no other case has been reported so far in which the mutation is leading to a normal tissue oxygen tension at a Hb level lower than normal. Finally, from our case, we can conclude that in patients with chronic anemia of unknown origin (rare anemia of complex mechanism), the study of Hb–O2 affinity has to be taken into consideration, because, in some cases, it may avoid the need for an expensive and invasive patient's workup. Moreover, during perinatal period, an awareness of this condition among physicians can spare newborns and their families from unnecessary evaluation and anxiety. This study has been approved by ethics committee of Hospital Clinic in Barcelona (Reference number 2010/4715) and was performed in the context of the Catalan Network for Diagnosis and Follow up of major haemoglobinopaties (CATGLOBIN, www.catglobin.cat). Authors are indebted to the connecting platforms of the EC co-financed European Network for Rare and Congenital Anaemias (ENERCA, www.enerca.org) and to the the PI14/00772 project, integrated in the National Plan R+D+I and co-funding by the ISCIII-Subdirección General de Evaluación and European Regional Development Fund (ERDF).