American Society of Hematology

Fig. 2.

$Fig. 2. Erythroid colony assays, mutation analysis, and Epo-dose response. / (A) Erythroid colony formation assays in 2 family members. The vertical axis indicates the numbers of BFU-E–derived colonies per 2.5 × 105 peripheral blood mononuclear cells, expressed as the mean ± SD of assays performed in triplicate. The final Epo concentration added to the cultures is indicated on the horizontal axis. (B) DNA sequence of subcloned genomic PCR products from the EpoR gene of the proband. Analysis of individual clones shows the sequences for the normal allele (left) and the mutant allele (right). The bold letter T indicates 5881G>T substitution resulting in introduction of a STOP codon. (C) Upper panel shows diagram of exon 8 of EPORgene. The coding sequence is shown as solid box and the 3′ untranslated region as an open box. The position and size of genomic PCR amplification products and a Tru9I (T) restriction map of the 493-base pair (bp) PCR amplification product for the mutant allele are shown. Lower panel shows detection of the mutation by restriction endonuclease digestion of PCR-amplified genomic DNA. Part of the pedigree is shown at the top of the figure. Genomic DNA amplification products were digested with Tru9I and fractionated by electrophoresis in a 2% agarose gel. The mutation creates a uniqueTru9I site in mutant allele and yields fragments of 369 bp and 124 bp in addition to the 493-bp fragment from the normal allele in individuals heterozygous for the new mutation. None of the unaffected individuals (open symbols) have Tru9I site in their genomic DNA, whereas all affected individuals in the family (filled symbols) are heterozygous for the mutation. (D) Epo dose-response of 32D cells transfected with wild type (WT) or mutant (ME) EpoRs. The cells were cultured in the indicated concentrations of Epo (U/mL) and the results are expressed as a percentage of maximal proliferation in 10 U/mL Epo as determined by MTT assay. Each data point represents the mean of 4 determinations with SE bars shown. Similar results were obtained in several experiments using multiple independent single cell clones of transfected cells.$

Erythroid colony assays, mutation analysis, and Epo-dose response.

(A) Erythroid colony formation assays in 2 family members. The vertical axis indicates the numbers of BFU-E–derived colonies per 2.5 × 10⁵ peripheral blood mononuclear cells, expressed as the mean ± SD of assays performed in triplicate. The final Epo concentration added to the cultures is indicated on the horizontal axis. (B) DNA sequence of subcloned genomic PCR products from the EpoR gene of the proband. Analysis of individual clones shows the sequences for the normal allele (left) and the mutant allele (right). The bold letter T indicates 5881G>T substitution resulting in introduction of a STOP codon. (C) Upper panel shows diagram of exon 8 of EPORgene. The coding sequence is shown as solid box and the 3′ untranslated region as an open box. The position and size of genomic PCR amplification products and a Tru9I (T) restriction map of the 493-base pair (bp) PCR amplification product for the mutant allele are shown. Lower panel shows detection of the mutation by restriction endonuclease digestion of PCR-amplified genomic DNA. Part of the pedigree is shown at the top of the figure. Genomic DNA amplification products were digested with Tru9I and fractionated by electrophoresis in a 2% agarose gel. The mutation creates a uniqueTru9I site in mutant allele and yields fragments of 369 bp and 124 bp in addition to the 493-bp fragment from the normal allele in individuals heterozygous for the new mutation. None of the unaffected individuals (open symbols) have Tru9I site in their genomic DNA, whereas all affected individuals in the family (filled symbols) are heterozygous for the mutation. (D) Epo dose-response of 32D cells transfected with wild type (WT) or mutant (ME) EpoRs. The cells were cultured in the indicated concentrations of Epo (U/mL) and the results are expressed as a percentage of maximal proliferation in 10 U/mL Epo as determined by MTT assay. Each data point represents the mean of 4 determinations with SE bars shown. Similar results were obtained in several experiments using multiple independent single cell clones of transfected cells.

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