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.

低氧亲和血红蛋白 (Hb) 突变是外周血氧饱和度 (SpO ₂ )低和无缺氧临床证据的贫血患者的一个罕见但重要的考虑因素；也可能出现紫绀。影响氧亲和力的 Hb 突变改变了参与 Hb 的松弛 (R) 氧合状态和紧张 (T) 脱氧状态之间的构象转换的关键分子区域。¹稳定 T 状态或损害 R 状态的突变会导致氧亲和力降低，并且氧解离曲线右移，p50 增加，此时 Hb 被氧饱和二分之一。血红蛋白浓度低于正常水平是由于向组织提供良好的氧气输送导致的红细胞生成驱动减少，并且红细胞生成素水平通常降低或正常。^{1, 2}低氧血症、贫血、有时出现紫绀的新生儿中存在 α (α)-珠蛋白​​基因的低氧亲和力突变，而 β (β)-珠蛋白​​基因的突变在约 6 个月大后出现归因于延迟 β -珠蛋白表达与胎儿 Hb 到成人 Hb 的出生后转换。高氧亲和力和低氧亲和力 Hb 均以常染色体显性方式遗传；还可以看到从头突变。¹

我们介绍了一名出生时SpO ₂测量值正常但在 22 个月大时发现持续严重低氧血症和轻度贫血的女童，此前未检测到；父母的血液学参数正常。她接受了广泛的心肺疾病评估，包括心导管检查和支气管镜检查，并接受了 1 年多的补充氧气治疗。在排除低氧血症的心肺病因后，患者接受了低氧亲和力 Hb 的评估。

这位女性在妊娠 39 3/7 周时通过自​​然阴道分娩出生于白人、非近亲、健康的父母。在出生第 3 天，她的双手和双脚的SpO ₂测量值均正常（≥98%）。她没有临床黄疸，看起来很正常。州新生儿血红蛋白病筛查通过等电聚焦报告胎儿和成人 (FA) Hb。在她出生住院期间和随后的常规随访中没有进行其他实验室检查；她表现出正常的生长发育，没有明显的紫绀。在 22 个月大时，她出现了上呼吸道感染的症状，并发现 SpO ₂严重低氧61% 室内空气。在因毛细支气管炎住院 10 天期间，虽然注意到呼吸道症状的临床缓解，但她持续低氧血症且无法停止吸氧。她出院时需要持续吸氧，并在随访中注意到需要鼻插管0.5 至 3 LO ₂ /min 以维持 SpO ₂  > 90%。

在接下来的 10 个月中，由于不明原因的低氧血症持续存在，进行了广泛的肺和心脏检查。她的肺部评估包括胸部计算机断层扫描 (CT)、软性支气管镜检查和腹部超声以排除肝肺综合征。心脏评估包括心电图、带气泡研究的超声心动图和心导管插入术。这些研究排除了肺动脉高压、心内分流和动静脉畸形，但注意到肺静脉中的SO ₂低（30% FiO _{2 时}为 81%–86% ）。所有其他研究均正常。动脉血气显示动脉血氧饱和度 (SaO ₂ )低至82%，PaO ₂升高111 mm Hg，高铁血红蛋白血症和碳氧血红蛋白血症呈阴性。在心肺检查结果为阴性的情况下，她被发现患有轻度正细胞性贫血（Hb 10.1 g/dL，MCV 89 fL）并且考虑了低氧亲和力 Hb 变异的可能性后转诊进行血液学评估。

Hb 氧亲和力由静脉血样本估计，p50 由 pH、O ₂和 Hb/O ₂饱和度计算。我们使用先前报告的数学公式转换为 Microsoft Excel 程序来快速计算 p50（方法在线补充）。计算出的 p50 升高至 41.9 mm Hg（正常为 24-29 mm Hg），表明存在低氧亲和力 Hb 变异体。通过毛细管电泳 (CE) 和高效液相色谱 (HPLC) 进行的初步筛选 Hb 分级测试未能显示明显的异常峰。然而，在色谱图上发现了异常高且变宽的 Hb A ₂峰。Hb A ₂之间的差异峰值表明存在大约 10% 的变异 Hb，它在 HPLC 上与 Hb A ₂共洗脱，并通过 CE 与 Hb A 一起迁移（图 1A、B）。α-珠蛋白基因测序未见异常；β-珠蛋白（HBB）测序发现了一种新的HBB变异体：c.317T>A；p.Leu106His。六溴联苯孩子父母（使用短串联重复标记确认亲子关系）的测序结果为阴性，表明发生了从头突变。在加州大学旧金山分校贝尼奥夫儿童医院奥克兰进行了完整的 Hb 氧解离曲线，并证实了右移的 Hb 氧解离曲线（图 1C）。p50 低于静脉血的单点估计值；可能是由于运输延迟导致红细胞三磷酸腺苷 (ATP) 和 2,3-二磷酸甘油酸 (BPG) 减少。

先前报道了相同密码子（Hb Bellevue IV）的 β-珠蛋白突变并显示不稳定³；因此，我们评估了溶血的存在。她没有网织红细胞增多症，也没有任何溶血生化标志物（总胆红素 0.1 mg/dL，乳酸脱氢酶 (LDH) 309 U/L，结合珠蛋白 82 mg/dL）。无创快速呼气末一氧化碳法（ETCOc < 1.0 ppm）未检测到显着溶血。然而，阳性异丙醇稳定性测试和间接亨氏体染色结果表明这种新型 Hb 变体具有轻度不稳定性。基于没有可检测到的明显溶血，Hb 稳定性降低的临床影响被认为是微不足道的。

诊断成立后，停止吸氧。六个月后，她继续无症状；在室内空气中，基线 SpO ₂测量值约为 70%。停止补充氧气后，她的轻度实验室贫血有所改善（11.4 g/dL，而接受补充氧气时为 10.1 g/dL）。停止补充氧气后 6 个月的血清促红细胞生成素水平仍然低于正常值，为 4 mU/mL，证实组织氧输送充足且不存在组织缺氧。

我们报告了一种新的低氧亲和力 Hb 变体，它在婴儿后期偶然检测到严重低 SpO ₂，没有组织缺氧的临床证据。否则无法解释的低 SpO ₂导致侵入性心血管和肺部检查，包括心导管插入术和支气管镜检查，回顾起来是不必要的。考虑低氧亲和力 Hb 解释了低氧血症和贫血，最终导致诊断和停止补充氧疗。由于低氧亲和力 Hb 突变通常以常染色体显性方式遗传，因此父母双方均不存在疾病表型这一事实使诊断更具挑战性。由于无法通过 CE 和 HPLC 清楚地检测到这种 Hb 变异，因此对该患者血红蛋白病的实验室评估也变得复杂。在 HPLC 上，异常 Hb 与 Hb A ₂共流出，产生更高更宽的峰，在 CE 上它与 Hb A 共迁移。

这种临床表现的分子基础是一种新的 β-珠蛋白突变（HBB c.317T>A；p.Leu106His，我们建议将其命名为“圣乔治血红蛋白”），导致低氧亲和力 Hb，解释了持续性低氧血症和轻度贫血。这种变异以前既没有在医学文献中也没有在基因特异性和一般人群数据库（外显子组变异服务器，基因组聚合数据库）中报道过。然而，β-珠蛋白基因相同密码子的另一个变体，HBB：c.316C>T；p.Leu105Phe（血红蛋白南密尔沃基）是红细胞增多症，相关联的高氧亲和性突变^4,3和HBB：c.317T> C。; p.Leu105Pro (Hb Bellevue IV) 被描述为与显着溶血相关的不稳定变体。³我们的患者没有任何临床可检测的溶血；然而，基于阳性异丙醇稳定性测试和诱导的亨氏体染色证明了突变体 Hb 的轻度不稳定性。在 γ-珠蛋白 2 ( HBG2 ) 基因中描述了相同的氨基酸取代，该基因与HBB具有同源性，（ HBG2 :c.317T>A；p.Leu105His，Hb F-Brugine/Feldkirch）。这种 γ 链变体还具有导致新生儿低氧血症的低氧亲和力。⁵我们的分析，包括通过数字聚合酶链反应 (PCR) 确定野生型和突变型 β-珠蛋白等位基因的相等比例，排除了这种新突变的性状体嵌合现象，并表明这是一种新生种系突变。

一些低氧亲和力 Hb 变体的特点是 SpO ₂和 SaO ₂测量值一致降低；其他人具有不一致的 SpO ₂和 SaO ₂值（低 SpO ₂但正常 SaO ₂）。我们在此描述的突变显示出与低 SpO2 和 SaO ₂值一致的减少。先前已报道了三种低氧亲和力 Hb 变体，它们具有一致的低 SpO ₂和低 SaO ₂测量值；Hb Bassett（HBA2或HBA1：c.284A>C）、Hb Rothschild（HBB：c[112T>A 或 112T-C]）和 Hb Canebiere（HBB）:c.307A>C)。⁶已报告了更多 Hb 变体，具有低 SpO ₂测量值和正常 SaO ₂；表明人为的低 SpO ₂可能是由于在脉搏血氧仪发射的波长下 Hb 变体的异常吸收光谱。^2、6

低氧亲和力 Hb 变异体在临床上是良性的，因为组织氧合是正常的，不需要医学治疗。¹然而，如本例所示，在这些患者中检测到的异常氧饱和度和有时出现的紫绀可能导致对低氧血症病因进行广泛且可能有害的调查。本报告强调了在没有组织缺氧的临床证据时在低氧血症的初步鉴别诊断中考虑低氧亲和力 Hb 变异体的重要性，以避免昂贵且可能有害的心肺检查和补充氧气的管理。