Novel, de novo, beta‐globin variant with decreased oxygen affinity (<scp><i>HBB</i></scp>:c.<scp>317T</scp>&gt;A, “<i>Hemoglobin St. George</i>”) in a healthy child with low oxygen saturations and anemia
Authors: Jessica A. Meznarich, Anton Rets, Archana M. Agarwal, Robert D. Christensen, N. Scott Reading, Frans A. Kuypers, Josef T. Prchal
Journal: American Journal of Hematology (2021)

Low oxygen affinity hemoglobin (Hb) mutations are a rare but important consideration in patients presenting with low peripheral oxygen saturations (SpO2) and anemia without clinical evidence of hypoxia; cyanosis may also be present. Hb mutations that affect oxygen affinity alter critical molecular regions involved in the conformational transition between the relaxed (R) oxygenated, and the tense (T) deoxygenated state of Hb.1 Mutations that stabilize the T state or impair the R state result in lowered oxygen affinity, and a right-shifted oxygen-dissociation curve with increased p50, the point at which Hb is one-half saturated with oxygen. Lower than normal Hb concentration arises due to decreased erythropoietic drive from excellent oxygen delivery to tissues, and erythropoietin level is typically decreased or normal.1, 2 Low oxygen affinity mutations of the alpha (α)-globin gene present in neonates with hypoxemia, anemia, and sometimes cyanosis, whereas mutations of the beta (β)-globin gene present after about 6 months of age attributed to delayed β-globin expression with the postnatal switch of fetal to adult Hb. Both high and low oxygen affinity Hbs are inherited in an autosomal dominant manner; de novo mutations are also seen.1 We present a female child who had normal SpO2 measurements at birth but at 22 months of age was found to have persistent severe hypoxemia and mild anemia that was previously undetected; parents had normal hematological parameters. She underwent extensive evaluations for cardiopulmonary disease, including cardiac catheterization and bronchoscopy, and was treated with supplemental oxygen for over 1 year. Having ruled out cardiopulmonary etiologies of hypoxemia, the patient underwent evaluation for low oxygen affinity Hb. This female was born by spontaneous vaginal delivery at 39 3/7 weeks gestation to Caucasian, nonconsanguineous, healthy parents. On day 3 of life, she had normal SpO2 measurements (≥98%) in both hands and feet. She had no clinical jaundice and appeared normal. State newborn hemoglobinopathy screening reported fetal and adult (FA) Hb by isoelectric focusing. No other laboratory testing was performed during her birth hospitalization and in subsequent routine follow-up; she exhibited normal growth and development without apparent cyanosis. At 22 months old, she presented with symptoms of an upper respiratory tract infection and was found to be severely hypoxemic with SpO2 61% on room air. During a 10-day hospitalization for bronchiolitis, although noted to have a clinical resolution of respiratory symptoms, she was persistently hypoxemic and unable to wean from supplemental oxygen. She was discharged on continuous supplemental oxygen, and in follow-up was noted to require between 0.5 and 3 L O2/min by nasal cannula to maintain a SpO2 > 90%. Over the next 10 months due to the persistence of unexplained hypoxemia, an extensive pulmonary and cardiac workup was undertaken. Her pulmonary evaluation included computerized tomography (CT) of the chest, flexible bronchoscopy, and abdominal ultrasound to rule out hepatopulmonary syndrome. Cardiac evaluation included electrocardiogram, echocardiogram with bubble study, and cardiac catheterization. These studies ruled out pulmonary hypertension, intracardiac shunts, and arteriovenous malformations but noted low SO2 in the pulmonary veins (81%–86% at 30% FiO2). All other studies were normal. An arterial blood gas showed low arterial oxygen saturation (SaO2) of 82% with an elevated PaO2 of 111 mm Hg, and was negative for methemoglobinemia and carboxyhemoglobinemia. In the setting of negative cardiopulmonary work-up, she was referred for hematology evaluation after being found to have mild normocytic anemia (Hb 10.1 g/dL, MCV 89 fL) and the possibility of a low oxygen affinity Hb variant was entertained. Hb oxygen affinity was estimated from a venous blood sample and p50 calculated from pH, O2, and Hb/O2 saturation. We used a previously reported mathematical formula converted to Microsoft Excel program for rapid calculation of p50 (Methods-online supplement). The calculated p50 was elevated at 41.9 mm Hg (normal 24–29 mm Hg), suggesting presence of a low oxygen affinity Hb variant. The initial screening Hb fractionation tests by capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) failed to show distinct abnormal peaks. However, an unusually high and broadened Hb A2 peak was appreciated on the chromatogram. Discrepancy between the Hb A2 peak values was suggestive of a variant Hb measuring approximately 10%, which co-eluted with Hb A2 on HPLC and migrated together with Hb A by CE (Figure 1A,B). Sequencing of α-globin genes revealed no abnormalities; β-globin (HBB) sequencing discovered a novel variant HBB:c.317T>A; p.Leu106His. HBB sequencing of the child's parents (paternity confirmed using short tandem repeat markers) was negative, indicating a de novo mutation. Full Hb oxygen-dissociation curve was performed at UCSF Benioff Children's Hospital Oakland and confirmed a right-shifted Hb oxygen-dissociation curve (Figure 1C). The p50 was lower than the single point estimate from venous blood; possibly from shipping delay that may have resulted in decreased red cell adenosine triphosphate (ATP) and 2,3-bisphosphoglycerate (BPG). A β-globin mutation at the same codon (Hb Bellevue IV) was previously reported and shown to be unstable3; thus, we evaluated for the presence of hemolysis. She did not have reticulocytosis nor any biochemical markers for hemolysis (total bilirubin 0.1 mg/dL, lactate dehydrogenase (LDH) 309 U/L, haptoglobin 82 mg/dL). No significant hemolysis was detected by the noninvasive rapid exhaled end-tidal carbon monoxide method (ETCOc < 1.0 ppm). However, positive isopropanol stability test and indirect Heinz body stain results were indicative of mild instability of this novel Hb variant. Based on the absence of detectable overt hemolysis, the clinical impact of the decreased Hb stability was considered insignificant. After the diagnosis was established, supplemental oxygen was discontinued. Six months later, she continued to be asymptomatic; baseline SpO2 measurements are around 70% on room air. Her mild laboratory anemia improved after stopping supplemental oxygen (11.4 g/dL compared to 10.1 g/dL while receiving supplemental oxygen). Serum erythropoietin level 6 months after supplemental oxygen was stopped, which remained low-normal at 4 mU/mL, confirming adequate tissue oxygen delivery and absence of tissue hypoxia. We report a novel low oxygen affinity Hb variant that presented with severely low SpO2 detected incidentally in later infancy, without clinical evidence of tissue hypoxia. The otherwise unexplained low SpO2 led to invasive cardiovascular and pulmonary investigations, including cardiac catheterization and bronchoscopy that in retrospect were unnecessary. Consideration of low oxygen affinity Hb explaining the hypoxemia and anemia eventually led to the diagnosis and discontinuation of supplemental oxygen therapy. Since low oxygen affinity Hb mutations are typically inherited in an autosomal dominant manner, the fact that the disease phenotype was not present in either parent made the diagnosis more challenging. Laboratory evaluation of this patient's hemoglobinopathy was also complicated by inability to clearly detect this Hb variant by CE and HPLC. The abnormal Hb co-eluted close to Hb A2 on HPLC, producing a higher and wider peak, and on CE it co-migrated with Hb A. The molecular basis of this clinical presentation was a novel β-globin mutation (HBB c.317T>A; p.Leu106His, that we propose naming "Hemoglobin St. George"), causing low oxygen affinity Hb, explaining the persistent hypoxemia and mild anemia. This variant has been previously reported neither in the medical literature nor in the gene-specific and general population databases (Exome Variant Server, Genome Aggregation Database). However, another variant at the same codon of the β-globin gene, HBB:c.316C>T; p.Leu105Phe (Hb South Milwaukee) is a high-oxygen affinity mutation associated with erythrocytosis,4, 3 and HBB:c.317T>C; p.Leu105Pro (Hb Bellevue IV) has been described as an unstable variant associated with significant hemolysis.3 Our patient lacked any clinically detectable hemolysis; however, mild instability of the mutant Hb was demonstrated based on a positive isopropanol stability test and induced Heinz body stain. This same amino acid substitution has been described in the gamma-globin 2 (HBG2) gene, which shares homology with HBB, (HBG2:c.317T>A; p.Leu105His, Hb F-Brugine/Feldkirch). This gamma-chain variant also has low oxygen affinity causing neonatal hypoxemia.5 Our analyses, including determination of equal ratio of wild-type and mutant β-globin alleles by digital polymerase chain reaction (PCR), ruled out gonosomal mosaicism of this new mutation and suggests this occurred as a de novo germline mutation. Some low oxygen affinity Hb variants are characterized by a concordant reduction in SpO2 and SaO2 measurements; others have discrepant SpO2 and SaO2 values (low SpO2 but normal SaO2). The mutation we describe here showed a concordant reduction with low SpO2 and SaO2 values. Three low oxygen affinity Hb variants with documented concordantly low SpO2 and low SaO2 measurements have been previously reported; Hb Bassett (HBA2 or HBA1:c.284A>C), Hb Rothschild (HBB:c[112T>A or 112T-C]), and Hb Canebiere (HBB:c.307A>C).6 More Hb variants have been reported with low SpO2 measurements and normal SaO2; suggesting an artifactually low SpO2 possibly due to abnormal absorption spectra of the Hb variant at wavelengths emitted by the pulse oximeter.2, 6 Low oxygen affinity Hb variants are clinically benign as tissue oxygenation is normal and no medical treatment is needed.1 However, the abnormal oxygen saturations and sometimes cyanosis detected in these patients can lead to extensive and potentially harmful investigations into the etiology of hypoxemia, as illustrated in this case. This report highlights the importance of considering low oxygen affinity Hb variants in the initial differential diagnosis of hypoxemia when there is no clinical evidence of tissue hypoxia to avoid costly and potentially harmful cardiopulmonary studies and administration of supplementary oxygen. The authors have no conflicts of competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The data that support the findings of this study are available in the supplementary material of this article. Data S1. Supporting information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.