A new 308 A-G substitution in HBG2 in an acyanotic newborn: case report

Abstract

Background

Neonatal hypoxemia in absence of structural cardiopulmonary anomalies is a diagnostic challenge. Haemoglobin variants involving the gamma-globin gene are recognized causes of transient desaturation in the newborn period. We report a novel HBG2 mutation identified in a clinically stable, non-cyanotic neonate born to a mother with active SARS-CoV-2 infection, highlighting a previously unreported variant with potential functional consequences on haemoglobin-oxygen affinity.

Methods

A term female newborn was delivered vaginally to a 39-years-old mother with gestational diabetes, thrombocytopenia, and SARS-CoV-2 infection, confirmed by real-time PCR at the time of delivery. The infant exhibited no signs of respiratory distress and normal blood gas analysis, but pulse oximetry revealed persistent arterial oxygen saturation of 88% in room air. Supplemental oxygen therapy (FiO₂ 23%) was initiated, resulting in a gradual improvement in saturation levels. Clinical examination and imaging were unremarkable, in particular no cardiac disease was present on heart ultrasound. Laboratory findings showed normal hemoglobin, haematocrit, and erythrocyte morphology, with a slightly reduced reticulocyte count.

Results

Hemoglobin electrophoresis revealed elevated fetal hemoglobin (HbF) (56.6%) with the presence of an abnormal variant band. Molecular analysis identified a heterozygous missense variant in the HBG2 gene (NM_000184.3:c.308 A > G), classified as likely pathogenic according to ACMG/AMP guidelines. Additionally, a promoter region polymorphism (NM_000184.3:c.-211 C > T (rs7882144), associated with increased fetal hemoglobin expression, was detected. No other etiologies were identified. The clinical course was uneventful and oxygen saturation normalized by one month of age without further intervention.

Conclusions

This case expands the known spectrum of neonatal hemoglobinopathies by describing a novel gamma-globin variant associated with transient hypoxemia. In the absence of methemoglobinemia, cyanosis, or structural abnormalities, a diagnosis of functional hemoglobinopathy was supported. While no causal relationship with maternal SARS-CoV-2 infection was established, this case raises questions about possible effects of maternal infection on fetal gene expression. Comprehensive diagnostic evaluation, including molecular analysis, is essential in cases of unexplained neonatal desaturation to avoid misdiagnosis and provide accurate prognostic and genetic counseling.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13052-026-02275-w.

Introduction

Neonatal haemoglobin (Hb) abnormalities, including those resulting from globin-gene mutations, can pose diagnostic challenges due to the dynamic physiology of human Hb during the perinatal period [1]. Hb is a tetrameric protein consisting of two alpha (α) and two beta (β) globin chains, each binding one oxygen molecule through an associated heme prosthetic group. The α-like and β-like globin-gene loci are developmentally regulated [2].

In fetus and newborn, the predominant globins expressed are α and γ, which combine to form fetal haemoglobin (HbF) (α2γ2). After birth, there is a shift in globin expression from γ to β, resulting in the production of HbA (α2β2). The genes encoding the globin chains, including HBA1, HBA2 (α-globin), HBB (β-globin), and HBG2 (gamma-globin), exhibit genetic variability that can lead to a plethora of Hb variants.

This physiological transition, combined with the presence of rare structural variants or regulatory polymorphisms, may result in discordant oxygenation parameters, such as reduced oxygen saturation in the absence of significant respiratory or cardiovascular abnormalities, thereby complicating early diagnostic assessment.

Physiologically, significant mutations in the γ-globin genes (HBG1 or HBG2) can lead to symptoms such as transient cyanosis in otherwise healthy newborns. These symptoms tend to resolve gradually in the first months of life as HbF is replaced by HbA. Five known γ-globin mutants have been associated with neonatal cyanosis, namely HB Toms River, Fort Ripley, Osaka, Cincinnati, and Circleville, each named after the patient’s city of origin [3–8].

In addition to structural variants, polymorphisms within regulatory regions of γ-globin genes, such as promoter variants, are known to modulate fetal hemoglobin expression and may influence both the biochemical phenotype and the clinical presentation of these conditions.

In this report, we present a case of γ-globin (HBG2) mutation in an acyanotic newborn with reduced arterial oxygen saturation. The diagnostic work-up, prompted by an abnormal hemoglobin profile and unexplained hypoxemia, included a stepwise molecular approach that excluded α- and β-globin gene defects and ultimately identified a γ-globin variant associated with a characteristic alteration in HbF composition. This case is particularly noteworthy given its occurrence in a neonate born to mother with coronavirus disease 2019 (COVID-19). The coexistence of a structural γ-globin variant and a regulatory polymorphism affecting HbF expression provides a clinically relevant model of gene–phenotype interaction in the neonatal period.

Case presentation

A full-term female infant weighing 3,380 g, AGA (adequate for gestational age), was delivered vaginally by a 39–years-old woman. The mother had been diagnosed with gestational diabetes and thrombocytopenia during pregnancy. Additionally, she tested positive with real-time PCR for SARS-CoV-2 at the time of delivery.

However, the newborn appeared healthy at birth, Apgar score was 9 and 10 at 1 and 5 min, respectively, and tested negative for SARS-CoV-2. Immediately after birth, the pulse oximetry screening revealed low oxygen saturation levels (88% SaO₂), so she started oxygen therapy in incubator (FiO₂ 23%) to achieve a saturation level of 94%. The infant exhibited no signs of respiratory distress and normal blood gas analysis (pH 7.41, pCO₂ 31 mmHg), with polycythemia (Hb 24 g/dL, Hct 74%).

The physical examination revealed no cyanosis, no cardiac murmur, no tachypnea, no retractions, and not any pathological findings.

Heart ultrasound showed the absence of any cardiopathies, and chest x-ray and lung ultrasound were normal.

Laboratory data showed low reticulocyte count 38.4 × 10⁹/L (n.v. 50–100 × 10⁹/L). Erythrocyte morphologic features were unremarkable. Blood culture was negative and complete blood count revealed hemoglobin (16 g/dL) and haematocrit (48%). Mean corpuscular volume (96.1 fL) and peripheral blood smear were normal too.

Blood gas analysis was performed to further investigate the hypoxemic condition and demonstrated pH 7.40 (n.v. 7.35–7.45), partial pressure of carbon dioxide (pCO₂) 35 mmHg (n.v. 35–45 mmHg), bicarbonate levels (HCO₃⁻) 21.7 mmol/L (n.v. 22–26 mmol/L), partial pressure of oxygen (pO₂) 79 mmHg (n.v. 80–100 mmHg), and oxygen saturation (sO₂) of 79.2% (n.v. >94%), consistent with isolated hypoxemia in the absence of respiratory or metabolic acidosis.

This discrepancy between relatively preserved pO₂ and reduced oxygen saturation suggested an alteration in hemoglobin oxygen affinity or function.

On the third day of life, blood gas analysis showed elevated methemoglobin levels (MetHb 3%; n.v. <1.5%), which could not be explained by exposure to oxidizing drugs or other known acquired cause and remained stable in the following days, with persistent oxygen requirement.

This finding further strengthened the suspicion of an underlying hemoglobin disorder and suggested us to carry targeted investigations for congenital hemoglobinopathies.

Electrophoresis showed that total hemoglobin consisted of approximately 56.6%, HbF including a variant band, and 39.3% HbA. High-performance liquid chromatography and capillary electrophoresis confirmed an abnormal hemoglobin profile characterized by elevated HbF levels (24.8–40.0%) and the presence of a variant fraction, consistent with a structural alteration of γ-globin chains.

We performed also hemoglobin electrophoresis of both parents, which was normal.

Molecular analysis on genomic DNA extracted from whole blood samples collected in EDTA was performed as previously described [9]. A stepwise molecular approach was adopted. Initial screening of β-globin (HBB) and α-globin (HBA1 and HBA2) genes by PCR amplification and direct Sanger sequencing did not identify pathogenic variants, thereby excluding common forms of thalassemia. Subsequent analysis of γ-globin genes (HBG1 and HBG2) identified a heterozygous missense variant, NM_000184.3:c.308 A > G (p.Asn103Ser), in the HBG2 gene, which was confirmed by amplification refractory mutation system (ARMS) analysis.

The c.-211>CT (rs7882144) variant in the HBG2 promoter was also identified, which is a known modulator of γ-globin expression associated with increased fetal haemoglobin (HbF) levels.

Most mutations, polymorphisms, or variants in the HBG2 promoter (such as c.-211 C > T, rs7882144, identified in this patient) act as modifiers of fetal hemoglobin (HbF) expression and may influence the severity of other hemoglobinopathies.

In this case, the presence of “favorable” variants can be protective because elevated HbF can ameliorate the severity of β-hemoglobinopathies by reducing globin chain imbalance. In the present case, this variant may act as a phenotypic modifier, potentially helping to reduce the severity of the clinical findings.

Although initially classified as a variant of uncertain significance based on standard databases, the identified γ-globin variant was further evaluated according to ACMG/AMP criteria. It fulfilled PM2 (absence from population databases) and PM5 (novel missense variant at a residue with known pathogenic substitutions), and was supported by multiple in silico tools (PP3), leading to an overall classification consistent with likely pathogenicity.

Screening for mutations in the HBG1/HBG2 genes was carried out by PCR-based amplification of each gene exon and splice-site boundary regions, followed by Sanger sequencing on an automated DNA sequencer (ABI PRISM™ 3130 XL, Life Technologies, CA, USA). Confirmation was performed using amplification refractory mutation system (ARMS) procedures [10].

Variant pathogenicity assessment was conducted according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines, using a scaled point system: 0–5 for variants of uncertain significance (VUS), 6–9 for likely pathogenic (LP) variants, and ≥ 10 for pathogenic (P) variants [11]. Genetic testing was performed on the singleton, as relatives were not available for testing.

As several studies showed, genetic investigations can represent a valid support in the identification of new variants to explain heterogeneous or otherwise inexplicable clinical pictures [12–14].

Extensive testing for metabolic abnormalities was unrevealing. Chest radiography, echocardiography, and cerebral ultrasound were normal. No specific treatment other than supportive oxygen therapy was provided. Clinical course was unremarkable, and Hb oxygen saturation gradually improved from approximately 88–90% to more than 93–95%. The patient was discharged home at 1 month of life without supplemental oxygen, with a HB oxygen saturation consistently higher than 95% and good clinical conditions.

Discussion and conclusion

This case report presents a unique clinical scenario of an apparently healthy full-term female neonate who exhibited reduced arterial oxygen saturation shortly after birth. The neonate was born to a mother who tested positive for SARS-CoV-2 during pregnancy.

The oxygen dependence of the newborn in the absence of cyanosis can have various etiologies. If an infant is hypoxemic or shows signs of respiratory distress but does not present cyanosis, this usually indicates poor oxygen-carrying capacity, hemoglobin affinity, or tissue perfusion rather than a simple ventilation problem. The differential diagnosis is between haematological causes, cardiological and haemodynamic causes and respiratory causes.

As we said, we excluded any cardiovascular and hemodynamic causes by echocardiography. Respiratory causes, as congenital anomalies of the airways, were excluded by chest X-ray and ultrasound, which were found to be normal.

Moreover, the not-cyanotic and not-tachypnoeic presentation suggested that the etiology may not be primarily related to cardiac or pulmonary abnormalities. Instead, it directed our attention toward haematological and/or genetic factors. In particular, the discrepancy between reduced oxygen saturation and the absence of respiratory or hemodynamic compromise raised the suspicion of an intrinsic hemoglobin dysfunction affecting oxygen affinity or transport.

Genetic evaluation revealed a previously undescribed γ-globin gene mutation, NM_000184.3: c.308 A > G, p.Asn103Ser, classified as likely pathogenic according to ACMG criteria (PM2, PM5) and predicted deleterious by multiple in silico tools (AlphaMissense 0.74; PROVEAN − 4.5; CADD 25; REVEL 0.88; DANN 1.00; Sift4G 0.0010; MetaRNN 0.95; MetaSVM 1.0; MutationAssessor 4.3; PP3_strong), achieving a total of 8 ACMG points.

Notably, although initial laboratory classification suggested a variant of uncertain significance, the integration of molecular findings with the clinical phenotype and hematological profile supported its reclassification as likely pathogenic.

Moreover, a polymorphism localized in the promoter region of HBG2 (NM_000184.3:c.-211>T; rs7882144) was identified. Variants located in this regulatory region are well-established modulators of HbF expression [10]. By enhancing γ-globin transcription, these variants can contribute to increased HbF levels, which in turn may ameliorate the clinical severity of β-hemoglobinopathies, including sickle cell disease and β-thalassemia [15, 16].

In the present case, the coexistence of a structural γ-globin variant and a regulatory promoter polymorphism suggests a combined effect involving both qualitative and quantitative alterations of hemoglobin function, potentially modulating the observed phenotype.

The gamma gene is a pivotal component in the production of HbF, and alterations in this gene can exert profound effects on oxygen transport and tissue oxygenation. Notably, mutations in the γ-globin gene are relatively rare and primarily associated with neonatal cyanosis. Key clinical indicators encompass a positive family history and reduced Hb oxygen saturation in the absence of arterial hypoxemia in an infant who otherwise presents as healthy and exhibits no evidence of heart or lung disease [1, 7]. In our case, the presence of borderline pO₂ values in the context of significantly reduced oxygen saturation further supports a functional alteration of hemoglobin rather than a primary respiratory impairment. The rationale behind establishing this diagnosis lies in the importance of providing precise genetic and prognostic counselling. Such counsel aims to ensure adequate Xcardiac or pulmonary disorder [17].

Previously described γ-globin mutations have been associated with neonatal cyanosis, either by impeding the binding of oxygen to ferrous (Fe²⁺) Hb or by promoting its spontaneous oxidation (auto-oxidation) to ferric (Fe³⁺) methemoglobin, which is incapable of effectively binding oxygen [4, 5, 8]. These variants are collectively termed “M hemoglobins”. Distinct from the four other hemoglobin variants known to induce neonatal cyanosis, hemoglobin Toms River affects amino acid E11 of γ-globin and demonstrates a normal level of methemoglobin. Rees et al. [18] have summarized five HB variants characterized by aminoacid substitutions at the E11 position in β-globin, leading to either cyanosis or haemolytic anemia. The modest but significant increase in methemoglobin levels observed in our patient further supports the hypothesis of an altered redox behavior of the hemoglobin molecule, potentially related to the identified γ-globin variant.

Another important question to evaluate is the potential role of the maternal SARS-CoV-2 infection during pregnancy. While the neonate tested negative for COVID-19 and appeared healthy at birth with strong Apgar scores, the maternal COVID-19 diagnosis raises questions about potential fetal exposure and its impact on neonatal health.

There is currently no sure evidence linking SARS-CoV-2 infection to specific genetic mutations in newborns. However, it is important to remember the effect of epigenetic factors on genetic function, which can also be influenced by infections, including SARS-CoV-2, or by other factors such as gestational diabetes, nutritional or pharmacological agents.

However, rather than implying a direct mutagenic effect, accumulating evidence underscores the role of epigenetic mechanisms as key mediators between environmental exposures and gene expression during fetal development. Maternal factors such as SARS CoV 2 infection, gestational diabetes as observed in this case, nutritional status, and pharmacological exposures may influence gene regulation through DNA methylation, histone modifications, and non-coding RNA activity, without altering the underlying DNA sequence. These processes may contribute to phenotypic variability and plausibly interact with underlying genetic variants, including the γ globin mutation identified in this patient, thereby modulating their clinical expression. Consistent with this view, similar gene environment interactions have been reported in pediatric disorders in which epigenetic modulation shapes the phenotypic expression of genetic variants [19].

Taken together, the integration of clinical findings, hemoglobin analysis, and molecular data supports a coherent pathophysiological model in which a γ-globin structural variant, potentially modulated by a promoter polymorphism and environmental influences, leads to altered oxygen affinity and transient neonatal hypoxemia.

Further research is needed to establish whether a potential interplay exists between maternal viral infections and neonatal genetic mutations. Moreover, this case report underscores the critical importance of conducting comprehensive evaluations and genetic testing in neonates with atypical clinical presentations, as such efforts may lead to the identification of genetic mutations.

In conclusion, this case report underscores the complexity of neonatal healthcare, the significance of maternal health, and the diagnostic challenges associated with rare genetic variants. It serves as a reminder of the importance of thorough evaluation and consideration of all potential factors in neonatal cases with unusual clinical presentations, ultimately leading to improved patient outcomes.

Electronic Supplementary Material

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Abbreviations

Hb

Haemoglobin

HbF

Fetal hemoglobin

HPFH

Hereditary persistence of fetal hemoglobin

HbA2

α-globin

HbB

β-globin

HbG2

Gamma-globin

Hct

Hematocrit

AGA

Adequate for Gestational Age

SARS-Cov-2

Acute Severe Coronavirus 2 Respiratory Syndrome

Author contributions

S. P. and M. C. drafted the initial manuscript and revised the manuscript; N. L. e A. I. made substantial contributions to the conception of the manuscript and interpretation of the data; I. A. and R. R. made genetic analyses; M. C., N. L. and A. I. revised the manuscript. The final version of the manuscript was critically revised and finally approved as submitted by all the authors.

Funding

This research received no external funding.

Data availability

Data will be made available on reasonable request.

Declarations

Ethical approval

Not applicable.

Consent to participate

Not applicable. NICU consent, signed by parents at hospital admission, allows to use any personal data in anonymous form for scientific reason or publication.

Consent for publication

Consent for publication was obtained from parents.

Competing interests

Not applicable.