Novel Mechanism of β0 Thalassemia: Missense Mutation of the Last Nucleotide of β Globin Exon 1 (G->C) Leads to Undetectable Transcript and Mutant Peptide.
Authors: Agarwal Neeraj , Mojica-Henshaw Mariluz P. , Kutlar Ferdane , Gaikwad Amos , Ou Ching N. , Kutlar Abdullah , Prchal Josef T. 
Published: October 2019

Abstract
Hemoglobin Monroe (Hb Monroe) results from a point mutation (G->C) in the last nucleotide of β globin exon 1, which is also the penultimate nucleotide of codon 30 of the β globin mRNA (AGG->ACG/Arg->Thr). Hb Monroe was described seventeen years ago simultaneously by two groups: in an African American female with β thalassemia intermedia, wherein the thalassemia was thought to result from highly unstable peptide (Hemoglobin.1989;13:67); and in a North African female with compound β thalassemia (Proc Natl Acad Sci U S A.1989;86:1041) wherein the Hb Monroe mutation was thought to result in abnormal pre-mRNA splicing as detected in an in vitro cell-free transcription assay. We evaluated a 31-year old previously asymptomatic woman of Asian Indian (Bengali) descent, who presented with flu like symptoms and found to have low hemoglobin level (9.5 gm/dL), microcytosis (MCV 68 fL), moderately elevated liver enzymes and serum ferritin concentration of 3000 ng/ml. A liver biopsy revealed increased liver iron and significant fibrosis. Hemoglobin analysis, which was interpreted as compound heterozygosity for HbE/β0 thalassemia, revealed HbF: 51%, HbE: 43.2%, HbA2: 5.8%. β globin gene sequencing showed Hb Monroe and E mutations. The asymptomatic brother of the proband had borderline anemia (Hb 12 gm %), microcytosis (MCV 70 fl), HbF: 8.5 %, HbA: 87.2%, HbA2: 5.0% and was heterozygous for Hb Monroe mutation by Bme 15801 restriction enzyme analysis of genomic DNA. We set out to determine the molecular basis of the thalassemia phenotype associated with Hb Monroe mutation and whether this mutation in our subjects is present on African haplotype or had arisen independently. Since we could not detect the mutant peptide either in fresh hemolysate or reticulocyte enriched preparations; we expanded the peripheral blood erythroid progenitor cells in vitro of both proband and her brother. Hemoglobin analysis by both HPLC and mass spectrophotometry did not detect Hb Monroe peptide in the expanded cells. β globin cDNA from the reticulocytes and expanded erythroid progenitors was amplified using three different primer sets and no splice variants were detectable. Sequencing of the amplified cDNA revealed only normal β globin mRNA transcript. Other β globin gene mutations cis to Hb Monroe are being ruled out; to date, we have not found any promoter region or stop codon mutations, deletions or splicing mutations from promoter - 90 region to 3′ UTR including poly-A region. Haplotype analysis revealed a different haplotype from the two African American patients, indicating an independent origin of Hb Monroe mutation in our cases. Interestingly, both of our cases have elevated HbF, as did the two originally reported Hb Monroe patients (16.5 and 85%) and a currently unreported African American patient with sickle cell - β0 thalassemia due to Hb Monroe (11.4%). Hb F was also high in a group of nine patients reported with sickle cell - β0 thalassemia due to Hb Monroe (3.1%– 8.9%; Hemoglobin.1998; 22:153). We conclude that this missense mutation, IVS1-1 (G->C/Arg 30 Thr) results in undetectable transcript and mutant Hb peptide leading to β0 thalassemia. The molecular mechanism of the undetectable mutant transcript is being investigated.