American Society of Hematology

Figure 3.

$Figure 3. Effects of the chromosome 12 abnormalities in 2 patients with PNH. (A) Structure of normal and abnormal HMGA2 locus in J20 and US1. White, black, and gray boxes indicate UTRs, coding regions, and abnormally fused fragments, respectively. The exon numbers of HMGA2 are shown below the boxes. The nucleotide sequences on both sides of BP2 and BP7 (arrowheads) are shown in the white and gray boxes. The truncated HMGA2 exon 5 of J20 and US1 are indicated by the brackets with the size (bp) shown above the brackets. The bent arrows indicate the fused fragments. The 3′ UTR of exon 5 of HMGA2 on long chromosome 12 is disrupted as a result of insertion of fragment b (the 12q12q14 fragment from short chromosome 12; see Figure 1). In the case of US1, a similar disruption of exon 5 resulted from the rearrangement that occurred when material deleted from 12q (fragments c and d) was inserted into 12p (see Figure 1). (B) Real-time PCR analysis of HMGA2 transcripts in J20 and US1. The amount of HMGA2 transcripts in bone marrow cells of 5 healthy individuals and of patients J20 and US1 were quantitated by using the TaqMan MGB PCR method. The positions of the forward (right-facing arrow) and reverse (left-facing arrow) PCR primers are indicated above the normal HMGA2 locus shown in panel A. The relative expression of HMGA2 transcripts is normalized to expression of β-glucuronidase transcripts. Each value of the relative expression indicates the average of triplicate measurements. Expression of HMGA2 was greater than normal (mean ± SD, 3.87 ± 0.45) for both J20 (mean, 10.23) and US1 (mean, 19.34) (*P < .01). The long and short horizontal bars indicate average and standard deviation (SD) in healthy individuals, respectively. (C) Allele-specific expression of HMGA2. A polymorphic region (based on TC repeats) in the 5′ UTR of HMGA2 was amplified by PCR and analyzed by polyacrylamide gel electrophoresis. The products were also cloned and sequenced to characterize the polymorphisms. (Top panel) A 183-bp product (containing 29 TC repeats) was generated from the J20-derived hybrid cell line containing long chromosome 12 (lane 1), whereas a 179-bp product (containing 27 TC repeats) was generated from the cell line containing short chromosome 12 (lane 2). Analysis of the PCR product generated by amplification of cDNA derived from GPI-AP– bone marrow cells of J20 revealed only the 183-bp product (lane 3). No PCR products were visualized when the PCR template was prepared without reverse transcriptase (lane 4). (Bottom panel) A 171-bp product (containing 23 TC repeats) was generated from the US1 hybrid cell line containing the der(12) (lane 5), whereas a 175-bp product (containing 25 TC repeats) resulted from amplification of DNA from the cell line containing normal chromosome 12 (lane 6). Analysis of the PCR product generated by amplification of cDNA derived from unfractionated bone marrow cells of US1 revealed only the 171-bp product (lane 7). No PCR products were visualized when the PCR template was prepared without reverse transcriptase (lane 8). The asterisk (left of each panel) indicates the position of an uncharacterized PCR product. The abnormal allele-specific expression of HMGA2 in the bone marrow cells of US1 was confirmed by using a genetic analyzer (3100-Avant; Applied Biosystems) (not shown). For both J20 and US1, HMGA2 expression appears to be derived exclusively from the mutant allele.$

Effects of the chromosome 12 abnormalities in 2 patients with PNH. (A) Structure of normal and abnormal HMGA2 locus in J20 and US1. White, black, and gray boxes indicate UTRs, coding regions, and abnormally fused fragments, respectively. The exon numbers of HMGA2 are shown below the boxes. The nucleotide sequences on both sides of BP2 and BP7 (arrowheads) are shown in the white and gray boxes. The truncated HMGA2 exon 5 of J20 and US1 are indicated by the brackets with the size (bp) shown above the brackets. The bent arrows indicate the fused fragments. The 3′ UTR of exon 5 of HMGA2 on long chromosome 12 is disrupted as a result of insertion of fragment b (the 12q12q14 fragment from short chromosome 12; see Figure 1). In the case of US1, a similar disruption of exon 5 resulted from the rearrangement that occurred when material deleted from 12q (fragments c and d) was inserted into 12p (see Figure 1). (B) Real-time PCR analysis of HMGA2 transcripts in J20 and US1. The amount of HMGA2 transcripts in bone marrow cells of 5 healthy individuals and of patients J20 and US1 were quantitated by using the TaqMan MGB PCR method. The positions of the forward (right-facing arrow) and reverse (left-facing arrow) PCR primers are indicated above the normal HMGA2 locus shown in panel A. The relative expression of HMGA2 transcripts is normalized to expression of β-glucuronidase transcripts. Each value of the relative expression indicates the average of triplicate measurements. Expression of HMGA2 was greater than normal (mean ± SD, 3.87 ± 0.45) for both J20 (mean, 10.23) and US1 (mean, 19.34) (*P < .01). The long and short horizontal bars indicate average and standard deviation (SD) in healthy individuals, respectively. (C) Allele-specific expression of HMGA2. A polymorphic region (based on TC repeats) in the 5′ UTR of HMGA2 was amplified by PCR and analyzed by polyacrylamide gel electrophoresis. The products were also cloned and sequenced to characterize the polymorphisms. (Top panel) A 183-bp product (containing 29 TC repeats) was generated from the J20-derived hybrid cell line containing long chromosome 12 (lane 1), whereas a 179-bp product (containing 27 TC repeats) was generated from the cell line containing short chromosome 12 (lane 2). Analysis of the PCR product generated by amplification of cDNA derived from GPI-AP^– bone marrow cells of J20 revealed only the 183-bp product (lane 3). No PCR products were visualized when the PCR template was prepared without reverse transcriptase (lane 4). (Bottom panel) A 171-bp product (containing 23 TC repeats) was generated from the US1 hybrid cell line containing the der(12) (lane 5), whereas a 175-bp product (containing 25 TC repeats) resulted from amplification of DNA from the cell line containing normal chromosome 12 (lane 6). Analysis of the PCR product generated by amplification of cDNA derived from unfractionated bone marrow cells of US1 revealed only the 171-bp product (lane 7). No PCR products were visualized when the PCR template was prepared without reverse transcriptase (lane 8). The asterisk (left of each panel) indicates the position of an uncharacterized PCR product. The abnormal allele-specific expression of HMGA2 in the bone marrow cells of US1 was confirmed by using a genetic analyzer (3100-Avant; Applied Biosystems) (not shown). For both J20 and US1, HMGA2 expression appears to be derived exclusively from the mutant allele.

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