Missense Mutation of the Last Nucleotide of Exon 1 of β-globin Gene Interferes with the Expression of Wild Allele.
Authors: Agarwal Neeraj , Kutlar Ferdane , Reading Scott , Kutlar Abdullah , Prchal Josef 
Published: April 2020

Abstract
A 31 y/o woman of Asian Indian descent was evaluated for high serum ferritin (&gt;3000 ng/ml), Hb 9.5 gm % and MCV 68 fL. A MRI revealed no increase in cardiac iron but hepatic siderosis. No mutations of HFE, ferroportin, and hemojuvelin genes were detected. HPLC revealed: HbF: 51%, HbE: 43.2%, HbA2: 5.8%. A compound heterozygosity for HbE/b0 thalassemia was suspected. b globin gene sequencing revealed HbE and a missense mutation (b globin IVS1 (−1) G-&gt;C) previously described as Hb Monroe. The asymptomatic brother of the proband had Hb 11.5 gm %, MCV 70 fL, HbF: 8.5%, HbA: 87.2%, HbA2: 5.0%, and heterozygosity for Hb Monroe mutation. We set out to determine the molecular basis of the thalassemia phenotype associated with Hb Monroe in the proband’s brother and in an African American family known to be heterozygous for Hb Monroe. Clinical and laboratory parameters of the study subjects are summarized in figure 1. β globin DNA and β globin cDNA from the reticulocytes and in vitro expanded erythroid progenitors was sequenced. β globin haplotype was analyzed. The quantity of β globin gene expression (18S RNA normalized) by real time quantitative PCR in study subjects was compared to the healthy controls. Previous studies on Hb Monroe invoked either unstable mutant peptide or aberrantly processed mRNA as the basis of thalassemia but none studied native mRNA. We did not detect mutant peptide or any splice variants. Sequencing of the amplified cDNA revealed only normal β globin mRNA. Further, no promoter region or stop codon mutations, deletions or splicing mutations were found from promoter - 90 region to 3′ UTR including poly-A region. Quantitative β globin expression assay revealed more than 70% reduction in β globin mRNA in Asian Indian subject as well as in two African American subjects albeit with different β globin haplotype (figure 2). Similar reduction of β globin mRNA with different β globin haplotypes rules out an unlikely possibility of an unidentified promoter mutation present in trans. Nonsense-mediated decay (NMD) is a cellular mechanism of mRNA surveillance which detects and degrades mRNA containing premature translation termination codon (PTC). Heterozygous cases of β0 and β+ thalassemia with PTC have been found to have reduction of β globin mRNA by 40% and 27% respectively, suggesting interference with transcription of mutant allele. However, we did not find PTC generating mutations either in the in vitro generated mRNA transcripts or in any theoretically formed transcripts created and analyzed in silico. Moreover, our subjects have more than 70% reduction of the β globin gene expression suggesting interference with transcription of not only mutant allele but also wild allele. We conclude that this missense mutation interferes with the expression of wild allele.
Figure 1: Clinical and laboratory parameters in our study subjects: A) Asian Indian (East Indian) female (proband), compound heterozygous for Hb Monroe and Hb E; B) Asian Indian (East Indian) male (proband’s brother), heterozygous for Hb Monroe; C) African American female #1, heterozygous for Hb Monroe; D) African American female #2, heterozygous for Hb Monroe. Figure 1:. Clinical and laboratory parameters in our study subjects: A) Asian Indian (East Indian) female (proband), compound heterozygous for Hb Monroe and Hb E; B) Asian Indian (East Indian) male (proband’s brother), heterozygous for Hb Monroe; C) African American female #1, heterozygous for Hb Monroe; D) African American female #2, heterozygous for Hb Monroe. Figure 2: β globin quantitative gene expression profile from subjects heterozygous for Hb Monroe mutation (relative percent expression compare to healthy controls): 1) Average value of three healthy individuals (100%), 2) Asian Indian male (21%), 3) African American female #1 (25%), 4) African American female #2(27%) Figure 2:. β globin quantitative gene expression profile from subjects heterozygous for Hb Monroe mutation (relative percent expression compare to healthy controls): 1) Average value of three healthy individuals (100%), 2) Asian Indian male (21%), 3) African American female #1 (25%), 4) African American female #2(27%)