Molecular basis of clonal expansion of hematopoiesis in 2 patients with paroxysmal nocturnal hemoglobinuria (PNH)

Abstract

Somatic mutation of PIGA in hematopoietic stem cells causes deficiency of glycosyl phosphatidylinositol-anchored proteins in paroxysmal nocturnal hemoglobinuria (PNH) that underlies the intravascular hemolysis but does not account for expansion of the PNH clone. Immune mechanisms may mediate clonal selection but appear insufficient to account for the clonal dominance necessary for PNH to become clinically apparent. Herein, we report 2 patients with PNH whose PIGA-mutant cells had a concurrent, acquired rearrangement of chromosome 12. In both cases, der(12) had a break within the 3' untranslated region of HMGA2, the architectural transcription factor gene deregulated in many benign mesenchymal tumors, that caused ectopic expression of HMGA2 in the bone marrow. These observations suggest that aberrant HMGA2 expression, in concert with mutant PIGA, accounts for clonal hematopoiesis in these 2 patients and suggest the concept of PNH as a benign tumor of the bone marrow.

Figure 1.

Chromosomal abnormalities in 2 patients with PNH. (A) Idiogram of normal chromosome 12 (Chr 12) modified from the NCBI Map viewer. Labeled gray boxes indicate the positions of the designated BAC clones used for FISH analysis. (B) J20. The karyotypic abnormality identified in the GPI-AP^– bone marrow cells of J20 was defined as an interchromosomal insertion. An 18.5M-bp region from q12 to q14 (surrounded by broken lines) is deleted in short chromosome 12 as defined by characterization of BP1. The 2.7-kbp small fragment (bracketed arrowhead, labeled a) and the 18.5-Mbp large fragment (bracketed rectangle, labeled b) deleted from short chromosome 12 are inserted inversely and directly, respectively, into the 12q14 region of long chromosome 12 generating BP2, BP3, and BP4. The deleted region into which the 2 fragments are inserted lacks 17 kbp of sequence (broken lines). BP1, BP2, BP3, and BP4 indicate the breakpoint junctions generated by the chromosomal abnormality. (C) US1. The karyotypic abnormality identified in the bone marrow cells of US1 was defined as an intrachromosomal insertion. The large fragment (19.5 Mbp, labeled c) and the small fragment (300 kbp, labeled d) are inserted into the TEL locus (gray arrowhead) on 12p13. BP5 is generated by the deleted region. BP6, BP7, and BP8 are generated by rearranged fragments c and d. (D) Sequences of BP junctions in J20 and US1. The sequences around BP junctions 1 to 8 are shown. BAC clones containing the sequence are denoted above the lines. Arrows indicate the nucleotide numbers of the BAC clones. Arrowheads indicate one of the candidate breakpoints, and gray regions indicate ambiguous sequences shared between the 2 BAC clones at the site of the breakpoint.

Figure 2.

Confirmation of breakpoint junctions generated by the chromosomal abnormalities. (A) Confirmation of the breakpoints in J20 by PCR. Genomic DNA from L1 (lane 1), L2 (lane 2), S1 (lane 3), S2 (lane 4), JY25 (the wild-type control) (lane 5), white blood cells (WBCs) from J20 (lane 6), WBCs of a healthy volunteer (lane 7), or no DNA template (negative control, lane 8) was used for PCR analysis using primer sets designed according to the flanking regions of BP1, BP2, and BP3 (illustrated in Figure 1). Both the integrity and quantity of the DNA templates were confirmed by PCR using control primers (labeled Control). That appropriate-sized PCR products were amplified using DNA derived from the circulating WBCs of J20 (lane 6) shows that both the S1 and L1 versions of chromosome 12 were present in vivo. (B) Confirmation of the breakpoints in US1 by PCR. Genomic DNA from WBCs of a healthy volunteer (lane 1), bone marrow cells of US1 (lane 2), PMN of US1 (lane 3), US1W (the hybrid cell line containing wild-type chromosome 12) (lane 4), or US1M [the hybrid cell line containing (der(12)] (lane 5), or no DNA template (negative control, lane 6) were used for PCR analysis using primer sets designed according to the sequence of flanking regions of the BP5, BP6, BP7, and BP8 (illustrated in Figure 1). These experiments show that both wild-type chromosome 12 and der(12) were present in the peripheral blood and bone marrow of US1. (C) Metaphase FISH showing the chromosomal abnormality in J20. BAC probes 471G7, 150C16, and 366L20 were hybridized against chromosomal specimens derived from the cell line (L1) that contains only long chromosome 12. Two hybridization signals (arrows) were detected with all 3 BAC probes, confirming that long chromosome 12 contained the inserted material deleted from short chromosome 12. (D) Metaphase FISH showing the chromosomal abnormality in US1. BAC probes 366L20, 438I19, and 474P2 (illustrated in Figure 1) were hybridized with chromosomal specimens derived from bone marrow cells of US1. Each sample contained a der(12) (indicated by 2 hybridization signals on the same chromosome, double arrows) and a wild-type chromosome 12 (indicated by one hybridization signal, arrow). The intrachromosomal insertion splits the signal on 12p generated by hybridization of probe 438l19, whereas signals are generated on 12q and 12p when probes that overlap BP5 on the centromeric (474P2) and telomeric (366L20) ends are used.

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.