Deep sequencing of whole genome exon in paroxysmal nocturnal hemoglobinuria
Authors: Li‐Yan Li, Zhao‐Yun Liu, Hui Liu, Chun‐Yan Liu, Zong‐Hong Shao, Rong Fu
Journal: American Journal of Hematology (2017)

To the Editor: Paroxysmal nocturnal hemoglobinuria (PNH) is a very rare disease that has been investigated for over one century and has revealed unique aspects of the pathogenesis and pathophysiology of a hemolytic anemia, which results from expansion of a clone of hematopoietic cells that, as a consequence of an inactivating mutation of the X-linked gene PIG-A, are deficient in glycosylphosphatidylinositol (GPI)-linked proteins, but only PIG-A mutations did not occur PNH.1 Up to now, there are three kinds of theories on the PNH clonal proliferation mechanism: (1) "Immune escape" theory: GPI—cells escape immune attack, leading to the proliferation of PNH clones to gain advantage, (2) PIG-A gene mutation causes the GPI- cells to obtain the anti apoptotic characteristic. (3) Second gene mutation theory. In our study, we have used V2's NimbleGen capture probe to capture the whole exon group (WES), and performed whole genome sequencing of exon in 13 patients (seven from PNH patients and six from AA-PNH patients), which to examine the mutational history of PNH, and so as to further reveal the possible mechanism of PNH cloning to gain the advantage of proliferation. Seven patients with PNH (P group) and six patients with AA-PNH(A group) were diagnosed in the Hematology Department of General Hospital Tianjin Medical University from August 2015 to Mar 2016 according to international PNH Study Group Criteria.2 The clinical information and study platforms applied to each sample are described in Table 1. We performed WES for all 13 samples: We use PROVEAN (Protein Variation Effect Analyzer) software3 to predict the protein function of the amino acid change caused by the mutation, and get the evaluation of the catastrophe risk. Using the above methods, we selected 134 mutated genes from the sequencing results, and the protein structure prediction is harmful. The average numbers of pathogenic somatic SNVs and indels in coding regions were56 (range 46–63) in the P group and 42 in the A group (range 25–88).These coding variants tended to occur more frequently in the P group, although the difference was not significant (P = 0.6139). We detected somatic PIGA mutations (6 SNVs and 2 indels) in the PNH fractions of four of seven cases analyzed by WES. And we identified a total of four shear site, two truncation, and two frame-shift mutations in cases analyzed by WES. All mutations were grade one. We performed KEGG pathway enrichment analysis and GO gene annotation of 134 mutated genes screened, the union as a target gene set, there were 21 target genes, CUX1, SUZ12, RB1, MUC4, CTBP1, CBLB, CTF1, CACNB2, TCF7L2, MLL2, PML, LAMA4, COMP mutations were recurrent in PNH patients; ALPPL2, TNN, GP1BA, CTF1, MAGEC1, MITF, TET2, CTBP2 mutations were recurrent in AA-PNH patients (Figure 1). We also analysed the average numbers of pathogenic somatic SNVs and indels in coding regions, P group and A group were 34 and 13, respectively, there was no significant difference between the two groups (P = 0.206). Of the 177 candidate SNVs and indels, mutation of five genes (CUX1, TET2, MUC4, MLL2, and RB1) that relate to the clonal proliferation in malignant clonal disease, highly associated with myeloid neoplasms and solid tumor, which were shared by five of 13 patients (38.46%), these can regulate the proliferation and differentiation of many kinds of cells, and have tumor suppressor function, which can lead to abnormal proliferation of cells. Moreover, CUX1 gene can regulate the expression of autophagy genes, and GO analysis showed that the coding protein CDP has the functions of cell proliferation regulation, metabolism regulation, gene expression regulation, cell invasiveness, cell motility, cell apoptosis, extracellular matrix compositions, and so on.4, 5 Top panel represent the sequencing performed for each case and cell-of-origin subtypes. Each row represents a gene screened by PKGG pathway enrichment and GO gene annotation which belongs to known solid tumor associated genes or more frequently mutated genes in MDS or AML. Although it is unclear how a PNH clone expands in bone marrow, it is considered that the most important hypothesis implicates in PNH patient, additional clonal and subclonal mutations corroborate with the pathognomonic PIGA mutation responsible for the prevailing cell phenotype. Bone marrow failure causes cytopenias associated with a proliferative decrease of its hematopoietic stem cells and appears to be related to a pre-leukemic state. NGS projects were the realization of genetic heterogeneity due to a combination of somatic mutations and the complexity of clonal architecture reflective of the sequential acquisition of genetic defects.6 While general principles of clonal dynamics have been derived from the study of leukemia and other malignant tumors, they have not been explored in PNH. PNH is subject to clonal dynamics and selection forces similar to those observed in hematopoietic neoplasms. The mutational events discovered in our research are not unique to PNH, but overlap significantly with the spectrum of mutations seen in myeloid neoplasms and other solid tumor. Many of these mutations, or other mutations in the same genes, have been identified as key drivers of clonal evolution in myeloid neoplasms or cancer, including TET2, SUZ12, CUX1, MUC4, MLL2, and RB1 genes. While additional mutations have been described in PNH, our results illustrate that genetic clonal selection and evolution may be operative not only in malignant tumors, but also in benign hematologic diseases. In some cases, permissive leukemogenic effects appeared to be instigated by mutations in CUX1, TET2, MLL2, or MUC4, suggesting that these primary events arose, conveying an initial growth advantage, with a PIGA mutation as a subclone conveying an additional growth advantage. PNH clones with multiple mutations were present at substantially higher frequencies than those of clones with only mutations in PIGA. This finding implies that the presence of additional mutations confers an intrinsic growth advantage for PIGA-mutant cells. The clonal dominance in PNH has previously been speculated to occur due to either an intrinsic growth advantage of PIGA mutant cells (the "relative growth advantage hypothesis") and/or to cell-extrinsic factors that might provide a relative growth advantage for PIGA-mutant cells (the so called "immune escape hypothesis"). The "relative growth advantage hypothesis" posits that the PIGA mutation alone is essential, but not sufficient, to cause PNH. Consistent with this hypothesis, rare PIGA mutations have been found in a very small proportion of HSCs in healthy individuals who do not have PNH. At the same time, the fact that some PNH patients have been found to harbor several different PIGA mutations simultaneously supports the notion that the presence of PIGA mutations alone underlies clonal advantage. The mechanisms leading to the clonal expansion and dominance of PNH stem cells remain a topic of continued investigation. Our results suggest that future investigations into PNH will offer the opportunity to examine the implications of hematopoiesis sustained primarily by one or two mutated stem cells, with many cases demonstrating an accumulation of additional mutations that may be due to an increase in the self-renewal burden placed on stem cells as a consequence of the disease. In addition, there were a significant number of cases in which PIGA was the lone mutation, although WES data suggest that this may be less frequent when entire exomes are analyzed. In conclusion, we explored the genetic characteristics of PNH and AA-PNH, and found mutation of genes involved in proliferation and apoptosis, metabolic reprogramming with activated mitochondrial oxidative phosphorylation, and abnormal activation of signaling pathways. Our results reveal that PNH, a disease considered to be benign, shares genetic alterations and clonal composition similar to those of overtly malignant myeloid leukemias. Although these results must be validated in a larger numbers of samples, our results provide information that may be useful in revealing the possible mechanism of the proliferation of PNH clones and developing therapeutic strategies for PNH.