Congenital Methemoglobinemia via the CYB5R3 Gene

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
1714 CYB5R3$750.00 81479 Add to Order
Targeted Testing

For ordering targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 18 days.

Clinical Sensitivity

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).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 CYB5R3$990.00 81479 Add to Order
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Turnaround Time

The great majority of tests are completed within 20 days.

Clinical Features

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).

Testing Strategy

This test involves bidirectional Sanger sequencing using genomic DNA of all coding exons of CYB5R3 gene plus ~10 bp of flanking non-coding DNA on each side. We will also sequence any single exon (Test #100) or pair of exons (Test #200) in family members of patients with known mutations or to confirm research results.

Indications for Test

Candidates for testing include cyanosis in the presence of normal arterial pO2, co-oximeter reading with peak absorbance at 631nm, and dark red, chocolate, or brownish to blue blood color. Co-oximeter readings suggesting methemoglobinemia are confirmed by Evelyn-Malloy method. It should be noted that these tests do not distinguish between inherited and acquired forms of methemoglobinemia (Percy and Lappin 2008).


Official Gene Symbol OMIM ID
CYB5R3 613213
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT


Name Inheritance OMIM ID
Methemoglobinemia 250800


Genetic Counselors
  • Ewenczyk C, Leroux A, Roubergue A, Laugel V, Afenjar A, Saudubray JM, Beauvais P, Villemeur TB de, Vidailhet M, Roze E. 2008. Recessive hereditary methaemoglobinaemia, type II: delineation of the clinical spectrum. Brain 131: 760–761. PubMed ID: 18202104
  • Human Gene Mutation Database (Bio-base).
  • Kugler W, Pekrun A, Laspe P, Erdlenbruch B, Lakomek M. 2001. Molecular basis of recessive congenital methemoglobinemia, types I and II: Exon skipping and three novel missense mutations in the NADH-cytochrome b5 reductase (diaphorase 1) gene. Human mutation 17: 348–348. PubMed ID: 11295830
  • Percy MJ, Lappin TR. 2008. Recessive congenital methaemoglobinaemia: cytochrome b 5 reductase deficiency. British Journal of Haematology 0: 080305033838221–??? PubMed ID: 18318771
  • Singh S, Sethi N, Pandith S, Ramesh G. 2014. Dapsone-induced methemoglobinemia: “Saturation gap”-The key to diagnosis. Journal of Anaesthesiology Clinical Pharmacology 30: 86. PubMed ID: 24574600
  • Vallurupalli S, Manchanda. 2011. Risk of acquired methemoglobinemia with different topical anesthetics during endoscopic procedures. Local and Regional Anesthesia 25. PubMed ID: 22915889
  • Warang PP, Kedar PS, Shanmukaiah C, Ghosh K, Colah RB. 2015. Clinical spectrum and molecular basis of recessive congenital methemoglobinemia in India. Clinical Genetics 87: 62–67. PubMed ID: 24266649
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Bi-Directional Sanger Sequencing

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (  As required, DNA is extracted from the patient specimen.  PCR is used to amplify the indicated exons plus additional flanking non-coding sequence.  After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions.  In nearly all cases, the full coding region of each exon as well as 10 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of February 2018, we compared 26.8 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 14 years of our lab operation we have Sanger sequenced roughly 14,300 PCR amplicons. Only one error has been identified, and this was an error in analysis of sequence data.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 10 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants, due for example to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

Deletion/Duplication Testing via Array Comparative Genomic Hybridization

Test Procedure

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.

Analytical Validity

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.

Analytical Limitations

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.

Order Kits

Ordering Options

myPrevent - Online Ordering
  • The test can be added to your online orders in the Summary and Pricing section.
  • Once the test has been added log in to myPrevent to fill out an online requisition form.
  • A completed requisition form must accompany all specimens.
  • Billing information along with specimen and shipping instructions are within the requisition form.
  • All testing must be ordered by a qualified healthcare provider.


(Delivery accepted Monday - Saturday)

  • Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
  • For small babies, we require a minimum of 1 ml of blood.
  • Only one blood tube is required for multiple tests.
  • Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
  • During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
  • In cold weather, include an unfrozen ice pack in the shipping container as insulation.
  • At room temperature, blood specimen is stable for up to 48 hours.
  • If refrigerated, blood specimen is stable for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.


(Delivery accepted Monday - Saturday)

  • Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
  • For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
  • DNA may be shipped at room temperature.
  • Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
  • We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.


(Delivery preferred Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Culture and send at least two T25 flasks of confluent cells.
  • Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
  • Send specimens in insulated, shatterproof container overnight.
  • Cell cultures may be shipped at room temperature or refrigerated.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We strongly recommend maintaining a local back-up culture. We do not culture cells.
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