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Validation of a recombinant DNA construct ([micro]LCR and full-length [beta]-globin gene) for quantification of human [beta]-globin expression: application to mutations in the promoter, intronic, and 5'- and 3'-untranslated regions of the human [beta]-globin gene.

[beta]-Thalassemia is characterized by the reduced production of [beta]-globin chains as a result of mutations in the [beta]-globin gene (1). This reduction is predictable when mutations occur in the coding sequence, but not when they occur in the 5'- and 3'-untranslated regions (UTRs), the locus control region (LCR), the promoter, or the introns. Whether such mutations are involved in the reduction of the [beta]-globin chain production or are simple polymorphisms cannot always be inferred from clinical data. Transient transfection studies with a [beta]-globin promoter and an heterologous reporter gene have shown that promoter mutations can decrease transcription (2) and are then associated with the [beta]-thalassemia phenotype, as illustrated by the -30T [right arrow] A mutation (3). However, such studies have often failed to provide clear-cut data regarding the transcriptional effect of a mutation or a deletion occurring in a noncoding sequence (4), and quantitative data are lacking.

To bypass these limitations and to mimic as closely as possible the regulatory mechanisms of [beta]-human globin gene expression in vivo, we created a construct (pBLG), in which the entire human [beta]-globin gene was cloned behind the [beta]-[micro]LCR. Whereas previous assays used constructs bearing HS2 as a single LCR enhancer element (5, 6), we used the entire [beta]-[micro]LCR because it has been shown that the other three HS elements play also a key role in [beta]-globin transcription (7-11).

Nucleotides changes in various untranscribed or untranslated parts of the [beta]-globin gene representing thalassemic mutations or deletions were introduced in the construct. All the mutations assessed in our study were found in members of proband families presenting with [beta]-thalassemia or were created by directed mutagenesis. In addition to the wild type, variant pBLG constructs carrying the following mutations were generated: -101C [right arrow] T, +20C [right arrow] T, IVS-I-108T [right arrow] C, and IVS-I-110G [right arrow] A mutations (12-16); +10 (-T), +4043, and +1565 [right arrow] 1577 deletions (17-19); and two novel mutations (-223T [right arrow] C, and -42C [right arrow] G). The -30T [right arrow] A thalassemic mutation (3) was included as a control. The constructs were expressed in stably transfected mouse erythroleukemia (MEL) cells, and the amount of human [beta]-globin mRNA was measured in total RNA extracted from transfected MEL cells grown for 72 h in the presence of 5 mmol/L hexamethylene bisacetamide, a chemical inducer of erythroid differentiation (20).

Quantification was performed by competitive reverse transcription-PCR, using synthetic calibrator obtained by directed mutagenesis and in vitro transcription. Reverse transcription and coamplification of mRNA extracted from transfected MEL cells and calibrator RNA were performed in the same tubes with various ratios of target to calibrator templates during cDNA synthesis and increasing numbers of PCR cycles during the exponential phase of amplification. After separation on an agarose gel, both DNA bands were photographed and quantified by image analysis software. Target mRNA copy number was calculated based on the number of copies generated in the exponential phase of the PCR.

Data collected were analyzed with the Generalized Linear Model program of SPSS Win 10.0[TM] (SPSS Inc.). For the wild-type and mutated or deleted constructs, the copy number of human [beta]-globin mRNA per nanogram of total RNA from MEL cells was submitted to ANOVA with three trial factors (21) to assess the variability across PCR cycles (the first trial factor with three PCR cycles as levels), across the amount of calibrator RNA (the second trial factor with six levels), and across transfections (the third trial factor with three experiments as levels).

Results are reported as the grand mean [+ or -] SD across the levels of each trial factor, with the P value of the F-test. The CV (%) was used to express the variability within each trial factor of each construct. The reliability coefficient of the transfection factor is reported as the overall interassay reproducibility. Contrasts between mean values of [beta]-globin mRNA copy numbers were performed with the Scheffe method (21). (Details on the materials and methods used are available in a supplemental file accompanying the online version of this Technical Brief at

Human [beta]-globin cDNA was consistently amplified from the wild-type construct (Fig. 1), as confirmed by sequence analysis the amplicon. Murine hemoglobin in different MEL cells cultured with hexamethylene bisacetamide as well as quantification of murine glyceraldehyde 3-phosphate dehydrogenase in total RNA were highly reproducible (see supplemental file for additional results). Quantitative data obtained with different constructs are shown in Table 1. Although expression of the -223T [right arrow] C construct did not differ from that of the wild type, mild to markedly reduced expression was observed with the other constructs. Accordingly, the novel -223T [right arrow] C mutation is a polymorphism as also confirmed by normal biological data in a single heterozygous patient. Despite "silent" phenotypic features (12, 22) and previous in vitro data suggesting a lack of binding activity (23, 24), the -101C [right arrow] T mutation in the distal CACCC box has drastically reduced expression, like the known -30T [right arrow] A mutation in the TATA box.

These apparently discrepant conclusions lead to several comments: (a) a previous functional assay (12) produced in vitro results similar to ours; (b) the human cellular line used to show the lack of binding activity also lacks [beta]-globin expression (25, 26), suggesting a lack of transcriptional factors essential for [beta]-globin expression; (c) several of these factors, such as the erythroid Kruppel-like factor, bind to CACCC and are active at a level that can not be detected by binding assays (27-29); (d) whereas mutations in the proximal CACCC box are considered to be more severe, some, like -92C [right arrow] T, can also be silent (30). Altogether, these data support our in vitro observations in favor of a transcriptional activity of the distal CACCC box rather than the alleged lack of such activity. The current discrepancy between in vitro and in vivo observations could rather be the consequence of a cosegregate mutation located in a negative transcriptional regulator, as suggested by other reports (31, 32).

The novel -42C [right arrow] G mutation has a very mild transcriptional effect. This mutation is located in the [beta]-globin direct-repeat element, a highly conserved element found in mammalian [beta]-globin promoters (33). This is the first report of a mutation in the human [beta]-globin direct-repeat element. The observed mild negative transcriptional effect in vitro correlates closely with previous experiments on single mutations in the mouse [beta]-globin direct-repeat element (33). In our patient, the phenotype observed with -42C [right arrow] G/IVS-I-(-1)G [right arrow] C is comparable to the phenotype reported with +33C [right arrow] G/codon 39C [right arrow] T mutations (28), whereas the IVS-I-(-1)G [right arrow] C or codon 39C [right arrow] T mutations are [beta]°-thalassemia mutations (34).

Several mutations or deletions found within the 5'-UTR are associated with [beta]-thalassemia (18), but are not always confirmed by transient transfection studies (4). Assessment of 5'-UTR mutations with this assay brought further insight in understanding of the 5'-UTR function and mechanisms of disease. We tested three 5'-UTR mutations. In vitro data with +10(-T) pinpoint the lack of transcriptional defect associated with this mutation. This reproduces previous observations and supports the hypothesis that a translational defect may reduce globin chain synthesis in +10(-T) heterozygotes (6). The +20C [right arrow] T mutation and +4043 deletion showed a twofold decrease in residual activity compared with the wild-type construct. The 5'-UTR +20C [right arrow] T mutation has been observed only in cis with the IVS-II-745(C [right arrow] G) mutation (5), as hypothesized several years ago (13). Likewise and to the same extent, the 5'-UTR deletion +4043 has also been shown to alter the [beta]-globin transcription, despite a lack of evidence from previous transient transfection assays (4).

In addition to those quantitative data, our cell culture expression system was also used to assess the alleged abnormal mRNA splicing of the new IVS-I-108T [right arrow] C mutation (15). Discrepant conclusions have indeed been drawn regarding the role of the IVS-I-108T [right arrow] C mutation (14, 15). We found no splicing abnormality in our study, whereas the well-known IVS-1-110G [right arrow] A mutation, used as a control, displayed abnormal splicing (16) as well as an unexpected retention of IVS-1 (see supplemental file for additional results). Sequence analysis of both PCR bands showed either the insertion of 19 intronic nucleotides, as described previously (16), or full IVS-1 retention. Quantitative data obtained with IVS-1-108T [right arrow] C showed decreased [beta]-globin expression, which is consistent with the observed thalassemic syndrome and pinpoints the potential role of a currently unknown cis-acting regulatory element in this intron. Current in vitro and phenotypic data highlight the role of IVS-I in [beta]-globin gene regulation and open the way to the characterization of as yet uncharacterized cis-acting regulatory elements in this region.


Functional effects of 3'-UTR mutations were also addressed with our assay. The 3'-UTR 13-bp deletion +1565 [right arrow] 1557, previously identified in the single heterozygous mother of a thalassemic Turkish child carrying a compound mutation (19), was assessed. The mother presented with a typical thalassemic trait, but no study was performed to assess the impact of this mutation on [beta]-globin transcription. In our assay, the deletion showed a strong negative transcriptional effect, consistent with the biological observation and confirming the importance of the 3'-UTR in the transcriptional regulation of the [beta]-globin gene.

In conclusion, the combination of a cell culture expression system and competitive reverse transcription-PCR, as described here, enables one to assess and quantify the expression of wild-type and mutated human [beta]-globin genes. A full range of mutations can be introduced within the human [beta]-globin gene. Taking advantage of unique or double restriction sites to introduce the mutation allows answers to whether a particular sequence change is a mutation or a silent polymorphism. Accurately measuring the quantitative effect of single nucleotide changes or deletions on [beta]-globin gene expression should therefore help to unravel the complex genotype-phenotype relationships in [beta]-thalassemia, especially in complex cases of thalassemia intermedia.

MEL cell lines and the pBluescript containing the human [beta]-globin [micro]LCR were kindly provided by Dr. F. Galacteros (Hopital Henri Mondor, Creteil, France). We thank F. Lemaigre, G. Rousseau (Institute of Cellular Pathology, Brussels, Belgium), B. Leth (Ludwig Institute for Cancer Research, Brussels, Belgium), and S. Loric (Hopital St Antoine, Paris, France) for reading the manuscript and for helpful comments. We thank Dr. J. Billiet (AZ Brugge, Brugge, Belgium) for providing some clinical data.


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Leonid M. Irenge, [1] Michel Heusterspreute, [1] Marianne Philippe, [2] Isabelle Derclaye, [1] Annie Robert, [3] and Jean-Luc Gala, [4] *

[1] Applied Molecular Technologies, Center for Human Genetics, Universite Catholique de Louvain, Clos-Chapelle-aux-Champs, 30-UCL/30.46, B-1200 Bruxelles, Belgium;

[2] Department of Biochemistry, Cliniques Universitaires Saint-Luc, Universite Catholique de Louvain, Avenue Hippocrate, 30, B-1200 Bruxelles, Belgium;

[3] Biostatistics and Epidemiology, Clos-Chapelle-aux-Champs, 30-UCL/30.34, Universite Catholique de Louvain, B-1200 Bruxelles, Belgium;

[4] Applied Molecular Technologies, Queen Astrid Military Hospital, Rue Bruyn, 2, B-1120 Bruxelles, Belgium;

* address correspondence to this author at: Applied Molecular Technologies, Center for Human Genetics, Clos-Chapelleaux-Champs, 30-UCL/30.46, B-1200 Brussels, Belgium; fax 32-2-764-3959, e-mail
Table 1. Human [beta]-globin mRNA copy number in the MEL cell line
stably transfected with various [beta]-globin variants: means and
CVs per trial factors.

Mutation Mean (SD) copy number Source of
 of target RNA/ng variability
 total RNA, x 105 (3 trial factors)

 Cycle PCR
Wild type 5.89 [+ or -] 0.07 Calibrator RNA
 Cycle PCR
-223T[right arrow]C 5.90 [+ or -] 0.10 Calibrator RNA
 Cycle PCR
-42C[right arrow]G 4.04 [+ or -] 0.05 Calibrator RNA
 Cycle PCR
+20C[right arrow]4T 2.97 [+ or -] 0.04 Calibrator RNA
 Cycle PCR
-30T[right arrow]A 0.662 [+ or -] 0.014 Calibrator RNA
 Cycle PCR
-101C[right arrow]T 0.622 [+ or -] 0.010 Calibrator RNA
 Cycle PCR
Del +40[right arrow]43 3.14 [+ or -] 0.07 Calibrator RNA
 Cycle PCR
Del +10 (-T) 5.62 [+ or -] 0.08 Calibrator RNA
 Cycle PCR
IVS-I-108T[right arrow]C 2.78 [+ or -] 0.04 Calibrator RNA
 Cycle PCR
Del +1565[right arrow]1577 2.10 [+ or -] 0.02 Calibrator RNA

Mutation SD, x [10.sup.5] CV, % F-test

Wild type 0.10 1.6 0.39
 0.08 1.3 0.95
 0.08 1.4 0.52
-223T[right arrow]C 0.15 2.5 0.38
 0.28 4.7 0.25
 0.03 0.56 0.75
-42C[right arrow]G 0.09 2.3 0.16
 0.41 10 <0.001
 0.12 2.9 0.05
+20C[right arrow]4T 0.12 4.0 0.01
 0.07 2.4 0.60
 0.07 2.5 0.16
-30T[right arrow]A 0.04 6.3 0.02
 0.10 15 <0.001
 0.04 5.4 0.05
-101C[right arrow]T 0.02 3.0 0.17
 0.09 14 0.001
 0.01 1.6 0.60
Del +40[right arrow]43 0.06 1.8 0.28
 0.23 7.3 <0.001
 0.32 10 <0.001
Del +10 (-T) 0.04 0.80 0.051
 0.28 5.2 <0.001
 0.08 1.3 0.001
IVS-I-108T[right arrow]C 0.04 1.4 0.33
 0.13 4.9 <0.001
 0.14 5.1 <0.001
Del +1565[right arrow]1577 0.01 0.20 0.99
 0.04 1.9 0.94
 0.11 5.4 0.02
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Title Annotation:Technical Briefs
Author:Irenge, Leonid M.; Heusterspreute, Michel; Philippe, Marianne; Derclaye, Isabelle; Robert, Annie; Ga
Publication:Clinical Chemistry
Date:Oct 1, 2002
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