Case report: neonatal cyanosis secondary to congenital methemoglobinemia, a cause to consider in newborn cyanosis

Introduction

Congenital methemoglobinemia (metHb) is a rare cause of neonatal cyanosis, resulting from the oxidation of ferrous iron in hemoglobin (Hb) to ferric iron in metHb. The acquired forms are more common, typically arising from exposure to oxidizing substances that increase metHb production; however, congenital forms are more prevalent during the neonatal period (1). We present a case of congenital metHb with a genetic mutation not previously reported in the literature, emphasizing the necessity of capillary blood gas analysis and genetic testing in these patients to rule out hereditary causes. We present this article in accordance with the CARE reporting checklist (available at https://tp.amegroups.com/article/view/10.21037/tp-2024-553/rc).

Case presentation

We present a case of a male born at term who exhibited cyanosis unresponsive to oxygen administration. The patient was a 3-kg term male with no remarkable maternal-obstetric history. He exhibited cyanosis without evidence of respiratory distress in the first hours of life. Oxygen saturation was maintained around 90% despite fraction of inspired oxygen 1 with high-flow oxygen. Pre- and post-ductal oxygen saturation were found to be equal, both maintained at 90%. The initial physical examination was normal apart from central and peripheral cyanosis, as well as a discrepancy between arterial oxygen pressure (PaO₂) and oxygen saturation measured by pulse oximetry. Blood gas analysis indicated elevated PaO₂ despite oxygen saturation remaining at 90%. The parents reported no family history of medical and hematological diseases.

Complementary studies were normal, including echocardiography, cerebral ultrasound, blood tests, blood cultures, cytomegalovirus (CMV) testing, glucose-6-phosphate dehydrogenase (G6PD) level and Hb electrophoresis. The only finding was a mild left anterior pneumothorax. After ruling out pulmonary, cardiological, and infectious causes, hematological diseases, such as metHb, were considered. The metHb level was 20% in co-oximetry. After considering the diagnosis of congenital metHb, high-flow oxygen support was discontinued following 3 days of treatment. The patient showed good progress, with normal oxygen pressure measured by co-oximetry without respiratory support, while maintaining a saturation of 90%.

Access to genetic testing is included in our public health system, and parents agreed after prior informed consent. The first genetic test performed on blood DNA from the patient was next generation sequencing (NGS) panel which analyzes the exonic regions of the following genes: ABCB6, ABCG5, ABCG8, ADA, AHSP, ALAS2, ATP11C, ATRX, BCL11A, BMP2, BMP6, C15orf41, CYB5A, CYB5R3, EGLN1, EPAS1, EPO, EPOR, FTH1, FTL, GAPDH, GCLC, GLRX5, GPX1, GSR, HAMP, HFE, HJV, HMOX1, HP, HSPA9, KCNN4, KIF23, LPIN2, MMACHC, PFKL, PFKM, PGD, PKLR, PRDX2, RHAG, SH2B3, SLC25A38, SLC2A1, SLC40A1, SLC4A1, TMPRSS6, TRF2, UGT1A1, VHL. NGS detected a compound heterozygous missense mutation in the cytochrome B5 reductase (CYB5R3) gene: c673C>T (p.Arg225Cys) and c977A>G (p.His326Arg), both considered pathogenic/probably pathogenic. Table 1 shows the NGS results. The presence of a compound heterozygous genotype in the CYB5R3 gene may explain the phenotype observed in the patient with congenital metHb, as it is a recessive condition. Erythrocyte enzyme determination confirmed a decrease in CYB5R3 activity: CYB5R3 activity 0.22 IU/gHb (ref. range 2.26–3.42 IU/gHb).

Subsequently, the study was extended with Sanger sequencing of exons 7 and 9 of the CYB5R3 gene. This test was performed on the blood DNA from patient and his progenitors for family genetic counseling. The patient had c.574C>T (p.Arg192Cys) and c.878A>G (p.His293Arg) mutation, validating the variants identified by NGS. The mother had c.574C>T (p.Arg192Cys) mutation, and the father had c.878A>G (p.His293Arg) mutation, both in a heterozygous state. Figure 1 shows the results of the Sanger on the patient and his parents. Both parents were healthy and asymptomatic, as they are carriers of the mutation in heterozygous form on only one allele of the gene. This family genetic counseling confirmed the patient’s compound heterozygosity (one mutation per allele). The prognosis of the patient was uncertain at the time of diagnosis, as it involved a novel mutation.

Figure 1 Sanger sequencing of the exon 7 and 9 of the CYB5R3 gene in the patient and his parents. (A) Patient sample. Sequencing of exon 7 shows heterozygosis, with one normal allele and one with amino acid exchange in codon 192. In exon 9 there is also heterozygosis, with one normal allele and one with amino acid exchange in codon 293. (B) Sample from the mother. The mother presents the mutation in heterozygosis in exon 7, with a normal allele and another with amino acid exchange in codon 192; the sequencing of exon 9 is normal. (C) Sample from the father. The father has the mutation in heterozygosis in exon 9, heterozygous, with one normal allele and one with amino acid exchange in codon 293; sequencing of exon 7 is normal. This image has been published with the consent of the patient’s parents.

The patient has progressed favorably without any additional interventions or adverse events. He has achieved appropriate physical and mental development, without neurological disorders. There has been a decrease in perioral cyanosis, with metHb levels dropping to 3%. Figure 2 illustrates the evolution of the metHb levels in relation to the patient’s age. Oxygen saturation reached normal levels (96%) by 2 years of age. The family was advised to avoid the early introduction of leafy green vegetables and exposure to certain medications, such as some anesthetics.

Figure 2 Evolution of the methemoglobin levels in relation to the patient’s age.

All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Helsinki Declaration (as revised in 2013). Informed consent was taken from the patient’s guardians for publication of this case report and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.

Discussion

This case report contributes to expanding the differential diagnosis of newborns with cyanosis unresponsive to oxygen and to increase the awareness of neonatologists and pediatricians about the importance of a correct interpretation of the blood gas analysis and co-oximetry. In these patients, it is necessary to consider congenital hematological conditions, such as metHb. Confirming the type of metHb through genetic studies facilitates family counselling and the discovery of new mutations with pathogenic value. Due to the rarity of this disease, it is difficult to publish case series. Although this publication is limited by being a case report of a single patient and the potential involvement of abnormalities in unexamined non-coding regions of CYB5R3, the hematological and genetic findings remain relevant.

MetHb is an oxidized form of Hb, resulting from the oxidation of divalent ferrous iron (Fe²⁺) to ferric iron (Fe³⁺). This ferric iron (Fe³⁺) contributes to oxidative stress, as metHb is incapable of binding and transporting oxygen to the tissues (1). In addition, it increases the oxygen affinity of other forms of Hb, resulting in a leftward shift of the Hb dissociation curve. This shift is associated with higher oxygen affinity and reduced oxygen release to the tissues (2). As a consequence, tissue hypoxia occurs without a decrease in Hb levels (3). Nicotinamide adenine dinucleotide (NADH) CYB5R is the enzyme responsible for reducing ferric iron (Fe³⁺) to ferrous iron (Fe²⁺), thereby converting metHb to Hb. There are two isoforms of CYB5R: the soluble form, which is present only in erythrocytes, is responsible for the reduction of metHb to Hb. The membrane form is located in the endoplasmic reticulum and mitochondrial membranes in cells across all tissues, where it performs various functions, including cholesterol biosynthesis and P450-mediated hydroxylation of steroids and antibiotics (4).

The normal metHb level is less than 1% (1,5). MetHb occurs when there is an imbalance between the production and metabolism of metHb, resulting in metHb levels exceeding 3% (6). The level of metHb in the blood correlates with symptoms; when metHb levels exceed 3%, cyanosis and hypoxemia can be detected using a pulse oximeter (7). Patients are typically asymptomatic with metHb levels less than 10%. In cases where metHb exceeds 10%, symptoms may include headaches, tachycardia, or mild dyspnea (8).

MetHb interferes with the accuracy of pulse oximetry. Consequently, it is common for these patients to exhibit a discrepancy between pulse oximetry oxygen saturation and their clinical condition or PaO₂ (8).

MetHb can be either congenital or acquired. The acquired form is more common, often resulting from the administration of anesthetic drugs or excessive intake of nitrites. Neonates have 50–60% of adult levels of CYB5R, reaching normal enzyme levels by 12 months of age (9). Therefore, they are prone to develop metHb with excessive dietary nitrite intake.

Congenital causes are more common during the neonatal period. Congenital metHb is a rare cause of neonatal cyanosis, with an estimated prevalence around 1:100,000 (5). There are two forms of congenital metHb. The first form arises from mutations in the globin genes and is inherited in an autosomal dominant manner, collectively referred to as HbM disease (8).

The second form results from a genetic alteration in the CYB5R3 gene, inherited in an autosomal recessive manner (OMIM 250800). Two types of recessively inherited congenital metHb due to mutations in CYB5R3 are distinguished: Type I presents a decrease in the soluble isoform, affecting only blood cells and following a benign course, with cyanosis as the only symptom. Type II involves a reduction in both the soluble and membrane forms of CYB5R, impacting all tissues. Patients with Type II exhibit alterations in lipid metabolism and neurological involvement. Both isoforms of CYB5R are produced by a single gene, known as CYB5R3 or DIA1, which consists of 9 exons and is located on chromosome 22q13-qter (1).

Hereditary congenital metHb type I is associated with genetic missense mutations that result in the substitution of one amino acid for another, as detected in our patient. This substitution decreases the stability of the enzyme and promotes an accelerated reduction in its activity. In contrast, hereditary congenital metHb type II is associated with nonsense mutations and deletions that cause alterations in splicing, leading to the appearance of premature stop codons or frameshift mutations, which ultimately result in the inactivation of the enzyme (4,10). Our patient has a compound heterozygous missense mutation in the CYB5R3 gene. The c.574C>T (p.Arg192Cys) mutation, inherited from the mother, located in exon 7 of the CYB5R3 gene, is classified as pathogenic or likely pathogenic. It has been identified as a cause of recessive congenital metHb type I in the Indian population, as reported in the article by Kedar et al. (10). In this article, the authors demonstrate that the c.574C>T (p.Arg192Cys) mutation leads to a structural abnormality associated with a mild loss of function of the CYB5R protein (10). It is noteworthy that the article by Kedar et al. pertains to an Indian population and the race of patient and mother is European Caucasian. Furthermore, the c.878A>G (p.His293Arg) mutation, inherited from the father, is not previously described is not described in the literature, rendering its significance uncertain, although there are indicators suggesting potential pathogenicity. This may explain the phenotype observed in the patient, characterized by neonatal cyanosis unresponsive to oxygen administration, secondary to congenital metHb.

A review of the literature from the past 15 years identified two cases of type II metHb caused by compound heterozygous mutations in CYB5R3 (11,12). Both cases exhibited more severe clinical manifestations than our patient, including intellectual disability and cyanosis, and required methylene blue treatment due to significantly elevated metHb levels. Recently, a case of type I metHb due to compound heterozygous missense mutations has been reported (13). Similar to our patient, this case presented with mild clinical features, including cyanosis without other symptoms, normal intelligence, as well as appropriate psychomotor development and anthropometric measures.

CYB5R3 contains two functional domains: the FAD binding domain (residues 45–151) and the NADH binding domain (residues 177–284) (14). Therefore, the new variant we described, c.878A>G (p.His293Arg), does not affect any of these domains.

Our patient exhibited a favourable clinical course, presenting only with cyanosis at birth and first months but he improved with age, alongside adequate psychomotor development and no neurological involvement at the age of 2 years. Therefore, we could conclude that the diagnosis is congenital metHb type I, in which only the isoform present in red blood cells is affected.

The key diagnostic test for metHb is blood co-oximetry. In hereditary cases, metHb levels of 10–30% are common. In cases of suspected CYB5R deficiency, measuring CYB5R3 activity is considered the gold standard test, with enzyme activity typically being less than 20%. Our patient had a CYB5R activity of 10% of the reference value [0.22 IU/gHb (ref. range, 2.26–3.42 IU/gHb)]. To confirm the diagnosis, DNA sequencing of the CYB5R3 gene is necessary (8).

The primary treatment for acquired metHb is to discontinue the offending agents. For metHb levels above 30% or symptomatic cases, methylene blue and ascorbic acid may be considered. In congenital metHb, patients often tolerate metHb levels of 30–40% well and may have compensatory polycythemia, remaining asymptomatic. However, they are at high risk of acute decompensation when exposed to oxidizing agents (15). In most cases diagnosed with genetic alteration in the CYB5R3 gene type 1, cyanosis does not require treatment and it is primarily an aesthetic issue (8). The maximum level of metHb in our patient was 20%, with cyanosis as the only symptom, so he did not require any treatment.

Conclusions

MetHb is a rare condition that should be considered in the differential diagnosis of cyanosis in pediatric patients. The acquired form is more common, while congenital forms are more prevalent in neonates. Hereditary causes include autosomal recessive mutations in the CYB5R3 gene, which is responsible for converting metHb to Hb. This case report presents type I metHb with increased metHb and cyanosis limited to the neonatal period. Genetic testing confirmed a previously unreported mutation and contributed to family counseling. MetHb should be suspected in patients with cyanosis and hypoxia without respiratory distress, no improvement with oxygen therapy, and normal PaO₂, after excluding more common causes such as respiratory, infectious, and cardiological conditions.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the CARE reporting checklist. Available at https://tp.amegroups.com/article/view/10.21037/tp-2024-553/rc

Peer Review File: Available at https://tp.amegroups.com/article/view/10.21037/tp-2024-553/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-2024-553/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Helsinki Declaration (as revised in 2013). Informed consent was taken from the patient’s guardians for publication of this case report and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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