Heterogeneity of Molecular Biology of Methemoglobinemia; Study of Eight Consecutive Patients.

Methemoglobinemia is a rare congenital or acquired disorder characterized by increased levels of oxidized hemoglobin in which the ferrous iron (Fe++) is in the ferric (Fe+++) state. Congenital methemoglobinemia can be due to mutations of a) NADH Cytochrome b5 reductase (b5R), either type I - deficiency restricted to RBCs; and causing cyanosis, or type II - deficiency in all cells;(fatal in childhood), b) globin genes or c) cytochrome b5. Eight consecutive methemoglobinemia patients had spectrometric methemoglobin analyses, b5R activity assay, and analysis of b5R mRNA and genomic DNA. Four patients had mutations of b5R gene; two were novel (see Table). Two had type I and 2 had type II b5R deficiency. One was a compound heterozygote and three were homozygotes; 2 were intronic and 3 were exonic mutations. The molecular consequences of both intronic mutations were characterized; we report a novel mechanism of markedly reduced b5R transcript causing clinical type II deficiency. We also show that in one patient with type II deficiency the diagnosis would be missed by measuring the erythrocyte b5R activity alone. Three patients had acquired toxic methemoglobinemia; all had normal b5R activity and no mutations involving the b5R gene. One patient had congenital methemoglobinemia caused by a beta globin mutation (Hb MHyde Park). We conclude that: a) congenital methemoglobinemia due to mutations of b5R gene is at least as common as acquired methemoglobinemia; b) contrary to previous reports, b5R heterozygosity is not a common predisposition for acquired methemoglobinemia; c) congenital methemoglobinemias due to globin mutations are rare; and d) screening by enzyme assay is not a reliable predictor of b5R deficiency. Summary of results. The mutations marked by * are novel. | Pt# | Diagnosis | Methemoglobin | Cell type and B5R activity | B5R gene mutation | Consequences of mutation and remarks | |:------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | --------------- | ------------- | ------------------------------ | -------------------------------------------------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ | | RCM - recessive congenital methemoglobinemia, EBV - EBV transformed lymphocytes, PLT - platelets, GNC - granulocytes, MNC - mononuclear cells, B5R gene mutations numbered as in NCBI accession [AY341030][1] | | 1 | Type I RCM | 12% | RBC-20% | 1.22236 G →A in exon 6 and 2.22249 G →A* (IVS 6+1) | 1.178 Alanine → Threonine and 2. Aberrant mRNA processing with 3 different transcripts: a) in-frame skipping of exon 6 b) all exons +46 bp of intron 6 c) skipped exon 6 with 179 bp of intron 5. No normal transcript from this chromosome. Normal expression by Real time qPCR | | 2 | Type I RCM | NA | RBC-25%PLT, GNC and MNC - 100% | 29951 C →T*in exon 9 | 258 Arginine → Tryptophan | | 3 | Type II RCM | 30% | RBC - 50%and EBV - 25% | 22526 T →C in exon 7 | 203 Cysteine → Arginine | | 4 | Type II RCM | 20% | RBC - 2% and EBV - 25% | 22163 A →C (IVS 5–2) | Aberrant mRNA processing with 4 different transcripts: a)in-frame skipping of exon 6, b) normal transcript c) 217 bp of intron 6 included d) entire intron 6 (463 bp) included. Reduced expression by Real time qPCR with only 7% of normal mRNA in the patient and 28% of normal mRNA in the heterozygote mother. | | 5 | Infant Toxic | 35% | RBC - 60% | None | One week of diarrheal illness. Needed several administrations of methylene blue | | 6 | Toxic | 12% | RBC - 100% | None | Lidocaine exposure | | 7 | Toxic | 44% | RBC - 100% | None | Unidentified toxin or infection | | 8 | Hb.M- Hyde Park | NA | RBC - 100% | Beta globin gene @ codon 92 C → T | Beta globin 92 Histidine →Tyrosine | [1]: /lookup/external-ref?link_type=GEN&access_num=AY341030&atom=%2Fbloodjournal%2F104%2F11%2F1587.atom