Homozygosity for the hyperunstable hemoglobin variant Hb Agrinio (HBA2:c.89T>C) leads to severe antenatal anemia: Eight new cases in three families

Alpha-thalassemia commonly results from deletions of one (alpha +) or both (alpha zero) of the duplicated α-globin genes on chromosome 16 and, less frequently, from point mutations involving mainly the predominantly expressed α2-globin gene (HBA2).1 Molecular defects generating unstable α-globin chain variants are part of this category of nondeletional α-thalassemia. Clinical heterogeneity of hemoglobin (Hb) H disease, the common form of clinically significant alpha thalassemia, is mainly related to the genetic background, nondeletional HbH disease being more severe than the deletional forms.1 In Hb Bart's hydrops foetalis syndrome (BHFS), which associates severe in utero anemia, tissue hypoxia, developmental abnormalities, heart failure, and hydrops fetalis, all α-globin genes are generally missing. Occasionally, hydrops fetalis can result from the inheritance of a nondeletional variant from one parent and an alpha zero thalassemia allele from the other. These so-called “HbH hydrops syndromes” were also described in patients homozygous or compound heterozygous for a severe nondeletional HBA2 variant.1 Hemoglobin Agrinio (HBA2:c.89T>C) is an α2-globin gene variant leading to a Leucine to Proline substitution at codon 30 of the α-globin chain. This very rare variant, first described in 1993, generates a highly unstable α-globin chain, undetectable at the protein level by routine electrophoretic and chromatographic Hb studies.2 Simple heterozygotes for Hb Agrinio have the phenotype of α-thalassemia trait and do not exhibit hemolytic anemia usually associated with unstable hemoglobins.2 Compound heterozygotes bearing Hb Agrinio and deletional or nondeletional α-thalassemia in trans develop Hb H disease with hypochromic microcytic anemia, hepatosplenomegaly but relatively low levels of Hb H.2, 3 Up to now, 11 Hb Agrinio homozygotes have been reported in literature, their clinical course ranging from moderate non-transfusion-dependent anemia to severe transfusion-dependent thalassemia (TDT).3-6 The unexpected neonatal death of a newborn homozygous for Hb Agrinio (II-2) lead us to evaluate the clinical course of all patients with this genotype born and/or treated in France. All the French laboratories involved in the molecular diagnosis of hereditary red blood cell (RBC) disorders were contacted to collect all the homozygous cases of Hb Agrinio. Standardized clinical and laboratory data, including Sanger sequencing of the alpha and beta globin genes, Multiple Ligation Probe Analysis (MLPA) (MRC Holland) for the globin gene cluster analysis, and next generation sequencing (NGS) analysis of genes involved in RBC disorders, were collected. All parents gave informed written consent for molecular analyses and their authorization for participating in this study. Eight patients with homozygous Hb Agrinio belonging to three unrelated families were diagnosed between 2006 and 2020 (Table 1). Two families (I and III) originated from Spain and were from the gypsy community. Consanguinity was present among both with no link between the two families. Parents of the third family (II) who originated from Bulgaria were not consanguineous. There was one in utero death (III-2) at 22 weeks gestational age (GA) and seven live births. Prematurity (GA < 37 weeks) was present in 5 out of the 7 newborns (I-1, I-2, I-3, II-1, III-3) and severe hypotrophy (birth weight < 3rd percentile) in 3 (I-3, II-1, II-2). The median Hb level at birth was 6.85 g/dl (range 3.80–12.00). The three most anemic patients at birth (I-2, I-3, II-2) died during the first hours of life with hydropic features in 2 of them. Patient II-2 who motivated this study presented with cardiomegaly, hepatomegaly, severe pulmonary hypertension, cutaneous extramedullary hematopoiesis (blueberry muffin syndrome), and Hb values of 6 g/dl. She was hypotrophic but not premature and her antenatal ultrasound monitoring performed at 32 weeks of GA was reported as normal. Her 7-year-old sister, previously diagnosed with homozygous Hb Agrinio, was regularly transfused but did not experience neonatal distress. The parents had declined the proposal of prenatal diagnosis. Urogenital abnormalities were present in two male patients (I-1 and III-1): hypospadias with ambiguous genitalia requiring surgical treatment in one and medically treated micropenis and undescended testis in the second. All four survivors, currently aged 7–14, are regularly transfused and are receiving iron chelation: patient III-3, diagnosed in the antenatal period, had in utero transfusions (TF) at 23 and 27 weeks, because of an accelerated middle cerebral artery peak systolic velocity of 52 cm/s and of a Hb value of 4.5 g/dl. Patients I-1, II-1, III-1 were occasionally transfused in the first years of life and started regular TF (>8/year) later in childhood (at 6, 6, and 7 years respectively). All patients were homozygous for the HBA2:c.89T>C variant, without any additional beta or alpha globin gene cluster anomaly at gene sequencing or MLPA and all six parents were heterozygous. NGS sequencing of genes involved in hereditary RBC disorders was performed in two patients from two different families and relevant variants were then checked in the other patients. In family III, a heterozygous variant (c.1516G>A; p.Val506Ile) of the PKLR gene was found in the most severely affected child (III-3). In family I, a heterozygous SEC23B gene variant (c.716A>G; p.Asp239Gly) was detected only in the least severe of the three family cases (I-1). Eleven Hb Agrinio homozygotes were reported between 1998 and 2017, with Greek, Cypriot, Spanish or Macedonian origins.3-6 Among the 10 patients with an available follow-up, 5 had TDT at last evaluation and 5 required occasional or no TF after splenectomy. No death was reported, except for a probable case of BHFS in a Greek series: molecular diagnosis was not performed on the neonate but subsequent DNA analyses of the parents showed that they were both heterozygous for Hb Agrinio.4 In our experience of eight new patients with homozygous Hb Agrinio, the clinical course was more severe than reported in literature, with a presentation ranging from severe nondeletional transfusion-dependent HbH disease to BHFS, including congenital urogenital abnormalities. Antenatal anemia was profound in most of the fetuses and one benefited from in utero TF as in classical BHFS.1 In utero or neonatal death concerned half of the eight patients and additional ones were suspected in family I history, even if not biologically confirmed. All four survivors became transfusion-dependent and are currently under iron chelation therapy. No hematopoietic stem cell transplantation was performed because of the lack of available human leukocyte antigen-identical family donors. To try to explain this up-to-now unreported clinical severity and intrafamilial variability, we searched for the presence of aggravating genetic factors. Of note, genetic modulators of Hb H or BHFS phenotypes located outside the globin gene loci are poorly defined.1 The hypothesis of an additional molecular anomaly was partially investigated by the analysis of genes implicated in hereditary RBC disorder by NGS. A variant in the gene coding for pyruvate kinase (PK) was detected in family I. This variant is classified as class 3 according to the ACMG guidelines (Variant of Uncertain Significance), with controversial opinion of submitters in the databases. A second variant, reported once in databases, was identified in the SEC23B gene involved in congenital dyserythropoietic anemia (CDA2) for family III. These variants were found each in only one subject, at the heterozygous state, whereas PK deficiency and CDA2 are both autosomal recessive disorders, with heterozygotes being usually unaffected. However, the PKLR variant, which concerned the most severely affected sibling, could be involved in di-or multi-genism in association with another RBC disorder. Thus no general conclusion could be drawn. Due to the rarity of Hb Agrinio, clinical descriptions are scarce in literature and previous reports are mostly biological ones. The unforeseen clinical severity in Hb Agrinio homozygosity in this series can be also explained by a more comprehensive clinical analysis of the index cases and siblings, allowing the antenatal period to be better described. In conclusion, homozygous forms of Hb Agrinio, like some other severe and/or unstable alpha2-thalassemia variants (Hbs Adana, Taybe, Constant-Spring, Quong-Sze, or polyadenylation signal variants), can be a cause of “HbH” hydrops fetalis. In couples at-risk for homozygous Hb Agrinio, genetic counseling, including prenatal diagnosis, should be considered. If the pregnancy is continued, close fetal ultrasound monitoring is indicated and in case of severe fetal anemia in utero TF should be performed. [Correction added on September 13, 2022, after first online publication: The term “TF” has been changed to “utero TF” in the preceding sentence.] Isabelle Thuret initiated the study. Claire Berger, Sandrine Baron-Joly, Yoann Huguenin, Aurélie Cantais, Sophie Brun, Philippe Joly, Cécile Ged, Muriel Giansily-Blaizot, and Catherine Badens provided clinical and/or biological information from patients. Sarah Szepetowski, Isabelle Thuret, Serge Pissard and Patricia Aguilar-Martinez reviewed the literature and wrote the manuscript; All authors contributed to drafting of the manuscript, critical revision of the article and approved the final version of the manuscript. There are no competing interests for any author. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. All parents gave informed written consent for molecular analyses and their authorization for participating in this study. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.