Congenital Methemoglobinemia via the CYB5R3 Gene

In normal individuals, hemoglobin can become oxidized to form methemoglobin which is unable to bind oxygen. Congenital methemoglobinemia is a disorder characterized by chronically elevated methemoglobin levels due to impaired reduction of methemoglobin back to hemoglobin. There are two forms of the disease. Type I disease individuals present with cyanosis, slate-blue skin color, due to decreased hemoglobin oxygen saturation. However, most people are asymptomatic with benign methemoglobinemia. Type II disease symptom onset occurs in infants and is characterized by cyanosis, severe developmental abnormalities including mental retardations and failure to thrive. Other symptoms may include neurologic problems such as microcephaly, opisthotonus, seizures, athetoid movements, and spastic quadriparesis resulting from abnormal lipid elongation and desaturation and damage to the central nervous system. About 10-15% of cases of congenital methemoglobinemia are type II (Ewenczyk et al. 2008; Percy and Lappin 2008).

Both type I and II methemoglobinemia are due to cytochrome B5 deficiency with type I deficiency being restricted to erythrocytes compared to type II which affects all cells. Type I mutations are associated with decreased stability of the cytochrome b5 reductase protein. In non-erythroid cells, both soluble and membrane bound forms are synthesized replacing the unstable form. Erythrocytes are unable to compensate via protein synthesis as they are anuclear (Percy and Lappin 2008; Warang et al. 2015). Methemoglobinemia may also be caused by Hemoglobin M disease through mutations in either the HBA1 or HBB genes. Acquired methemoglobinemia can result from drug and topical anesthetics including dapsone and benzocaine (Singh et al .2014; Vallurupalli and Manchanda 2011).

Type I and II methemoglobinemia are inherited in an autosomal recessive manner through mutations in the CYB5R3 gene. The CYB5R3 gene encodes cytochrome b5 reductase and is involved in transfer of electrons from NADH to cytochrome b5. There are several isoforms generated through use of alterative promoters of cytochrome b5 resulting in both soluble and membrane bound forms of the protein. However, these isoforms only differ at exon 1 where there have been no reports of causative variants for methemoglobinemia. 

In type I methemoglobinemia, cytochrome b5 reductase deficiency is limited to erythrocytes which are incapable of replenishing easily degraded, heat-liable mutated proteins. This results in an impaired ability to convert methemoglobin back to its oxygen binding form, hemoglobin. Type I mutations are predominantly missense and non-overlapping with type II methemoglobinemia (Ewenczyk et al. 2008; Warang et al. 2015).

Type II methemoglobinemia, cytochrome b5 reductase deficiency is throughout the body and occurs in the membrane bound isoform present on the endoplasmic reticulum and outer mitochondrial membrane. The membrane bound form of cytochrome b5 reductase plays important roles in fatty acid desaturation and drug metabolism. Type II mutations primarily include deletions, nonsense, and splice site alterations leading to loss of protein. In rarer cases, missense mutations have been reported in patients with type II disease (Kugler et al. 2001; Percy and Lappin 2008).

In patients with laboratory findings indicating decreased cytochrome b5 reductase activity in erythrocytes and whole blood, clinical sensitivity for detection of CYB5R3 mutations is >95% (Ewenczyk et al. 2008; Percy and Lappin 2008). Analytical sensitivity should be high because the vast majority of mutations reported are detected by the method. Only one gross deletion has been reported in a single case (Human Gene Mutation Database).

This test provides full coverage of all coding exons of the CYB5R3 gene plus 10 bases of flanking noncoding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. We define full coverage as >20X NGS reads or Sanger sequencing. PGnome panels typically provide slightly increased coverage over the PGxome equivalent. PGnome sequencing panels have the added benefit of additional analysis and reporting of deep intronic regions (where applicable).

Dependent on the sequencing backbone selected for this testing, discounted reflex testing to any other similar backbone-based test is available (i.e., PGxome panel to whole PGxome; PGnome panel to whole PGnome).