A case of paroxysmal nocturnal hemoglobinuria caused by a germline mutation and a somatic mutation in PIGT

Abstract Ceruloplasmin (CP) is a multicopper ferroxidase that oxidizes ferrous iron promoting the binding of ferric iron to transferrin. The secreted form of CP (sCP) produced mainly by the liver is essentially absent in patients with aceruloplasminemia, a rare type of hereditary iron overload with brain, liver and pancreatic siderosis. Alternative RNA splicing generates a form of CP that is anchored by glycosylphosphatidylinositol (GPI-CP) to the membrane of astrocytes and immune cells. GPI-CP has been reported to help stabilize ferroportin, the only known iron exporter in mammal cells, so we aimed to investigate whether ferroportin expression is abnormal in circulating blood cells in aceruloplasminemia and in paroxysmal nocturnal hemoglobinuria (PNH), a naturally-occurring human model of acquired deficiency of GPI-anchored proteins. Peripheral blood samples were collected from two patients with aceruloplasminemia with different mutations on the CP gene: CP c.2879-1 G>T (splice site mutation) and CP c.2756 T>C (missense mutation), both with undetectable levels of sCP (<0.02g/L), one patient with a large PNH clone (89.9% type III), and a healthy control. Immunophenotype was determined by incubation with fluorescent antibodies against GPI-CP, ferroportin, and known lineage surface markers (CD45, CD14, CD19, and HLA-DR), data acquisition on a FACS Canto equipment, and analysis with software FACS Diva. GPI-CP and ferroportin were only detectable in CD19+ lymphocytes and monocytes in all samples. We found no significant differences across subjects regarding lymphocytic expression of GPI-CP or ferroportin. In monocytes, the expressions of both proteins in aceruloplasminemia with CP c.2879-1 G>T were similar to those seen in the control. Nevertheless, monocytic expression of GPI-CP and ferroportin were significantly reduced in CP c.2756 T>C and PNH, when compared to the control. These data confirm previous observations that B lymphocytes and monocytes express GPI-CP and ferroportin, and concomitant reduction of both expressions in PNH and in CP c.2756 T>C support that GPI-CP fosters ferroportin stability on the cell membrane. We also show that, while germline mutations of the CP gene generally cause undetectable sCP, there is heterogeneity in GPI-CP expression, which may remain preserved, as observed in CP c.2879-1 G>T. Further studies are necessary to clarify why this splice site mutation would still allow GPI-anchoring, while the CP c.2756 T>C point mutation abrogates the ability to anchor GPI-CP. While the preservation of lymphocytic GPI-CP was not surprising in an essentially myeloid PNH clone, normal GPI-CP in B lymphocytes in aceruloplasminemia suggests there are lineage-specific differences in physiological expression of ceruloplasmin forms between B lymphocytes and monocytes, with possible implications to the importance of iron metabolism in immune responses. We also noticed that the CP c.2756 T>C patient with monocytic reduction ferroportin presented with slightly more intense anemia and microcytosis. This could result from lower expression of ferroportin in bone marrow macrophages, with impaired iron delivery to erythroblasts, in analogy to monocytes. Finally, acquired ferroportin deficiency in PNH monocytes implies that loss of GPI-anchored protein not only exposes these cells to lysis by complement, but also to intracellular iron retention, generation of reactive oxygen species and may be involved in the pathophysiology of PNH. In summary, our data show that heterogeneity in GPI-CP expression in B lymphocytes and monocytes results in differential expression of ferroportin in aceruloplasminemia and PNH, and future studies should aim at investigating the implications of dysregulated iron metabolism in immune cells. Disclosures Fertrin: Apopharma Inc.: Honoraria; Alexion Pharmaceuticals Brasil: Speakers Bureau.

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CDKN1A-RAB44 transcript fusion and activation in cancers

10.1101/111856 ◽

2017 ◽

Author(s):

Han Sun ◽

Michelle Nguyen ◽

William Mueller ◽

Zhuanfen Cheng ◽

Hong Zeng ◽

...

Keyword(s):

Somatic Mutation ◽

Splice Site ◽

Targeted Therapies ◽

Large Scale ◽

Kinase Inhibitor ◽

Splice Site Mutation ◽

Hereditary Diseases ◽

Transcriptional Level ◽

Site Mutation ◽

Fusion Event

Splicing contributes to gene regulation and protein diversity, while abnormal splicing underlies both hereditary diseases and cancers. Various mutations that disrupt splicing factors, exonic or intronic splicing enhancers or silencers, as well as splice sites, could be responsible for abnormal splicing. Characterization of abnormal splicing events is not only helpful for understanding the molecular processes linking mutations to disease phenotypes, but also provides promising targets for targeted therapies. In addition, CRISPR/Cas9 editing could be benefited once more attention is given to potential abnormal splicing outcomes other than off-target effects at the DNA level. Although large-scale multiplexed genome editing has been demonstrated in yeast, and has also been attempted for particular exons or genes in other eukaryotic cells to achieve saturation, in practice it is much more difficult to measure splicing consequences with genome-wide saturation editing in human cells. Instead, massive somatic mutations accumulated in cancer cohorts provide invaluable opportunities to study somatic mutation-associated splicing events. Abnormal splicing is not necessarily limited to single genes. Transcript fusion is a special form of abnormal splicing that connects two or more genes due to splicing on a transcriptional level (rather than chromosomal translocations such as BCR-ABL in chronic myeloid leukemia). It could arise from conventional splicing on read-through transcripts when the two genes are next to each other and on the same strand, or from trans-splicing when two genes are on different chromosomes, strands or far away - a few cases had been reported. However, it was found that these fusions not only occurred in tumors but also in normal tissues; there was limited investigation regarding how the fusion could happen, whether it be due to mutations or not, and what the downstream perturbations were. Here, in an effort to characterize somatic mutation-associated abnormal splicing (especially in its simplest form, exon skipping events), we identified over one hundred such events in various tumors, including those in MET, PTEN and TP53. Surprisingly, we detected a recurrent, but previously undescribed, tumor-specific transcript fusion event between the cyclin-dependent kinase inhibitor CDKN1A and the RAS oncogene family gene RAB44. By creating genome-edited cell lines, we demonstrate a causal relationship between splice-site mutations in CDKN1A and the fusion to the RAB44 transcript. We further provide evidence that the fusion arises from a readthrough transcript that escapes exosome-mediated degradation when the splice-site mutation occurred, and we show that the presence of the fusion transcript correlates with TP53 inactivation and CDK activation. The strong tissue specificity of RAB44 and the relatively high prevalence of this transcript fusion in multiple types of cancers warrants further study which could inform subclassifications of these cancers and the development of targeted therapies.

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