Phosphatidylinositol Glycan Anchor Biosynthesis Class Research Articles

Single-cell and machine learning approaches uncover intrinsic immune-evasion genes in the prognosis of hepatocellular carcinoma

Background and aimsHepatocellular carcinoma (HCC) is a tumor of high heterogeneity and complexity, which poses significant challenges to effective treatment and patient prognosis because of its immune evasion characteristics. To address these issues, single-cell technology and machine learning methods have emerged as a promising approach to identify genes associated with immune escape in HCC. This study aimed to develop a prognostic risk score model for HCC by identifying intrinsic immune-evasion genes (IIEGs) through single-cell technology and machine learning, providing insights into immune infiltration, enhancing predictive accuracy, and facilitating the development of more effective treatment strategies. Materials and methodsThe study utilized data from The Cancer Genome Atlas database to analyze gene expression profiles and clinical data related to intrinsic immune evasion in patients with HCC. Various tools, including the Human Protein Atlas, cBioPortal, single-cell analysis, machine learning, and Kaplan-Meier plot, were used to analyze IIEGs. Functional enrichment analysis was conducted to explore potential mechanisms. In addition, the abundance of infiltrating cells in the tumor microenvironment was investigated using single-sample gene set enrichment analysis, CIBERSORT, xCELL, and tumor immunophenotype algorithms. The expression of glycosylphosphatidylinositol anchor attachment 1 (GPAA1) was examined in the clinical sample of HCC by quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemical staining. ResultsUnivariate Cox analysis identified 63 IIEGs associated with the prognosis of HCC. Using random forest, least absolute shrinkage and selection operator regression analysis, and support vector machine, a risk score model consisting of six IIEGs (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), phosphatidylinositol glycan anchor biosynthesis class U (PIGU), endoplasmic reticulum membrane protein complex subunit 3 (EMC3), centrosomal protein 55 (CEP55), autophagy-related 10 (ATG10), and GPAA1) developed, which was validated using 10 pairs of HCC and adjacent non-cancerous samples. Based on the calculated median risk score, HCC samples were categorized into high- and low-risk groups. The Kaplan-Meier curve analysis showed that the high-risk group had a worse prognosis compared with the low-risk group. Time-dependent receiver operating characteristic analysis demonstrated the accurate predictive capability of the risk score model for HCC prognosis. Furthermore, immune infiltration analysis showed a positive correlation between the risk score model and 40 immune checkpoint genes as well as Th2 cells. ConclusionsA prognostic risk score model was formulated by six IIEG signatures and showed promise in predicting the prognosis of patients diagnosed with HCC. The utilization of the IIEG risk score as a novel prognostic index, together with its significance as a valuable biomarker for immunotherapy in HCC, provides benefit for patients with HCC in determining therapeutic strategies for clinical application.

Paroxysmal Nocturnal Hemoglobinuria: Biology and Treatment.

Paroxysmal nocturnal hemoglobinuria (PNH) is a nonmalignant clonal hematopoietic disorder characterized by the lack of glycosylphosphatidylinositol-anchored proteins (GPI-APs) as a consequence of somatic mutations in the phosphatidylinositol glycan anchor biosynthesis class A (PIGA) gene. Clinical manifestations of PNH are intravascular hemolysis, thrombophilia, and bone marrow failure. Treatment of PNH mainly relies on the use of complement-targeted therapy (C5 inhibitors), with the newest agents being explored against other factors involved in the complement cascade to alleviate unresolved intravascular hemolysis and extravascular hemolysis. This review summarizes the biology and current treatment strategies for PNH with the aim of reaching a general audience with an interest in hematologic disorders.

Asfotase alfa improved skeletal mineralization and fracture healing in a child with MCAHS.

Tissue non-specific alkaline phosphatase (TNSALP) is an enzyme that is tethered to the cell membrane by glycosylphosphatidylinositol (GPI) and converts inorganic pyrophosphate to inorganic phosphate. Inorganic phosphate combines with calcium to form hydroxyapatite, the main mineral in the skeleton. When TNSALP is defective, conversion of inorganic pyrophosphate to inorganic phosphate is impaired and the skeleton is at risk of under-mineralization. Phosphatidylinositol glycan anchor biosynthesis class N (PIGN) is one of more than 20 genes in the GPI-biosynthesis family. Pathogenic variants in PIGN have been identified in multiple congenital anomalies-hypotonia-seizures syndrome (OMIM 614080), although a metabolic bone disease or skeletal fragility phenotype has not been reported. We describe a female child with multiple congenital anomalies-hypotonia-seizures syndrome due to a compound heterozygous pathogenic variant in PIGN who sustained a low-trauma distal femur fracture at age 7.4years. We hypothesized that the GPI synthesis defect may result in metabolic bone disease from inadequate anchoring of TNSALP in bone and initiated asfotase alfa, a human bone-targeted recombinant TNSALP-Fc-deca-aspartate peptide, as it could bypass the PIGN genetic defect that possibly caused her skeletal fragility. Asfotase alfa was begun at 8.5years. Baseline X-rays revealed mild rachitic findings of wrists and knees, which resolved by 5months of treatment. Bone mineral density (BMD) assessed by dual-energy X-ray absorptiometry (DXA) showed mild improvement in spine, hip and total body less head after 16months of treatment, while radius declined. She sustained additional low trauma fractures at right tibia and left humeral neck at 11 and 15months into treatment, which healed quickly. Calcium, phosphorus, and parathyroid hormone levels have remained within the normal range over the 18months of treatment. For adverse effect, she experienced a rash and discomfort in the first week of treatment which resolved with ibuprofen and diphenhydramine. She also developed subcutaneous fat atrophy. Overall, in this child with a compound pathogenic variant in PIGN, off-label use of asfotase alfa has been generally well tolerated with minimal side effects and resolution of rickets, but she continues to remain skeletally fragile.

Uncovering the genetic basis of hyperphosphatasia with impaired intellectual development syndrome type 2: identification of a novel biallelic nonsense mutation in PIGO gene

Background: Glycosylphosphatidylinositol (GPI) is a glycolipid containing phosphatidylinositol related to the protein surfaces by covalent attachment. Inherited GPI deficiencies have various phenotypic chrematistics, which range from intellectual disability to dysmorphic features, epilepsy, and other severe anomalies. Methods: Molecular diagnosis was performed using whole exome sequencing (WES) followed by Sanger sequencing. Results: WES revealed a novel homozygous nonsense variant (c.250C>T; p.Gln84Ter) in the exon 2 of the phosphatidylinositol glycan anchor biosynthesis class Ogene that might explain the disease phenotype in the patient. Conclusion: This study will help in proper genetic counselling of the family and help in genotype-phenotype correlation in the future.

A Genome-Wide Association Study for Susceptibility to Axial Length in Highly Myopic Eyes.

The online version contains supplementary material available at 10.1007/s43657-022-00082-x.

Ginkgetin promotes proliferation and migration of Schwann cells via PIGF/p38 MAPK signaling pathway

The proliferation and migration of Schwann cells is pivotal to peripheral nerve injury (PNI) repair. Recent studies have revealed that Ginkgetin has neuroprotective effects. Hence, we focused on identifying whether Ginkgetin could regulate the proliferation and migration of Schwann cells, thereby contributing to the repair of PNI. Rat Schwann cells RSC96 were treated with different concentrations of Ginkgetin. Short hairpin RNA targeting phosphatidylinositol glycan anchor biosynthesis class F (shPIGF) was employed to investigate the effects of PIGF on Ginkgetin-induced RSC96 cells. Viability of RSC96 cells was estimated via cell counting kit-8 (CCK-8) assay and proliferation of the cells was assessed by 5-ethynyl-2′-deoxyuridine (EdU) assay. Migration was estimated via wound healing assay and invasion was evaluated through transwell assay. Western blot was employed to test the expressions of PIGF, protein-38 (p38), and phosphorylated p-38 (p-p38). Ginkgetin (50 or 100 μg/ml) increased the viability, proliferation, migration, and invasion of RSC96 cells, up-regulated PIGF expression and raised the ratio of p-p38/p38, which were all reversed by PIGF silencing. Ginkgetin promotes proliferation, migration, and invasion of Schwann cells via PIGF/p38 MAPK signaling pathway.

Rare genetic causes of meningitis and encephalitis.

Differential diagnosis of meningitis and encephalitis is often very challenging because it cannot be determined based on symptoms, and the diseases have various causes. This article explains rare genetic causes of meningitis and encephalitis. Autoinflammatory disorders include cryopyrin-associated periodic syndromes, familial Mediterranean fever, and tumor necrosis factor receptor-associated periodic syndrome. Furthermore, other genetic disorders, such as complement factor I deficiency, phosphatidylinositol glycan anchor biosynthesis class T mutation, and neuronal intranuclear inclusion disease, can present as meningitis and encephalitis.

SUZ12 participates in the proliferation of PNH clones by regulating histone H3K27me3 levels.

Paroxysmal nocturnal hemoglobinuria (PNH) is a disease involving hematopoietic stem cell membrane defects caused by acquired phosphatidylinositol glycan anchor biosynthesis class A (PIGA) mutations. In this study, 97 target genes were selected as a target gene panel and screened in 23 PNH patients via the sequencing of specific DNA target regions. Through functional analysis, we identified that suppressor-of-Zeste 12 (SUZ12) may be involved in the proliferation of PNH clones. mRNA and protein expression levels of SUZ12 and the trimethylation level of histone H3 at lysine 27 (H3K27) in CD59- peripheral blood leukocytes from PNH patients were higher than those in CD59+ cells from PNH patients and peripheral blood leukocytes from healthy controls. In addition, the relative expression of SUZ12 in PNH patients was positively correlated with Ret% and the proportion of PNH clones. When we knocked down SUZ12 expression in a PIGA knockdown THP-1 cell line (THP-1 KD cells), the trimethylation of histone H3K27(H3K27me3) and cell proliferation decreased, apoptosis increased, and cell cycle arrest occurred in G0/G1 phase. In conclusion, SUZ12 participates in the proliferation of PNH clones by regulating histone H3K27me3 levels. Our results may provide new therapeutic targets and possibilities for PNH patients.

PIGN spatiotemporally regulates the spindle assembly checkpoint proteins in leukemia transformation and progression

Phosphatidylinositol glycan anchor biosynthesis class N (PIGN) has been linked to the suppression of chromosomal instability. The spindle assembly checkpoint complex is responsible for proper chromosome segregation during mitosis to prevent chromosomal instability. In this study, the novel role of PIGN as a regulator of the spindle assembly checkpoint was unveiled in leukemic patient cells and cell lines. Transient downregulation or ablation of PIGN resulted in impaired mitotic checkpoint activation due to the dysregulated expression of spindle assembly checkpoint-related proteins including MAD1, MAD2, BUBR1, and MPS1. Moreover, ectopic overexpression of PIGN restored the expression of MAD2. PIGN regulated the spindle assembly checkpoint by forming a complex with the spindle assembly checkpoint proteins MAD1, MAD2, and the mitotic kinase MPS1. Thus, PIGN could play a vital role in the spindle assembly checkpoint to suppress chromosomal instability associated with leukemic transformation and progression.

Further delineation of PIGB-related early infantile epileptic encephalopathy

Pathogenic variants in phosphatidylinositol glycan anchor biosynthesis class B (PIGB) gene have been first described as the cause of early infantile epileptic encephalopathy 80 (EIEE-80) in 2019. This disorder, an inherited glycosylphosphatidylinositol deficiency, is associated with a complex neurologic phenotype, including developmental delay, early-onset epilepsy and peripheral neuropathy. We report on a 5 year-old girl born from consanguineous parents, manifesting severe global developmental delay with absent speech, mixed peripheral polyneuropathy, hypotonia, bilateral equino-varo-supinated-cavus foot, early-onset scoliosis, elevated serum alkaline phosphatase and a single episode of febrile status epilepticus. Hypomyelination was documented on brain MRI. Whole-exome sequencing (WES) disclosed the likely pathogenic biallelic PIGB NM_004855.4: c.463G > C, p.(Asp155His) missense variant. In our patient, while other characteristic clinical, neuroimaging and laboratory findings (as described in the first research paper) were present, seizures were not a major clinical issue, thus contributing to our knowledge on this ultra-rare disorder.

Upregulation of phosphatidylinositol glycan anchor biosynthesis class C is associated with unfavorable survival prognosis in patients with hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common types of malignant tumor, and is the second highest cause of cancer-associated mortality, behind lung carcinoma. It is urgent to identify novel genes that can be used to confirm the diagnosis and prognosis of patients with HCC. The present study aimed to investigate the expression pattern of phosphatidylinositol glycan anchor biosynthesis class C (PIGC) in HCC and assess its clinical prognostic significance. Bioinformatics analyses were used to investigate PIGC mRNA expression levels in HCC and adjacent non-cancerous tissue samples. Furthermore, the present study detected the expression levels of PIGC protein in HCC and matched normal tissue samples via immunohistochemistry, and evaluated the prognostic significance of PIGC protein in HCC. The levels of PIGC mRNA and protein were found to be significantly higher in tissue from patients with HCC compared with non-cancerous liver tissue. The survival analysis showed that the expression levels of PIGC mRNA or protein were associated with the survival of patients with HCC. PIGC protein expression was significantly associated with Tumor-Node-Metastasis stage. A negative correlation between PIGC DNA methylation and mRNA expression was observed (Spearman r=−0.453). PIGC is an oncogene that is negatively regulated by DNA methylation, and high levels of PIGC mRNA or protein may predict an unfavorable prognosis in patients with HCC.

Expanding the phenotype of PIGS-associated early onset epileptic developmental encephalopathy.

The phosphatidylinositol glycan anchor biosynthesis class S protein (PIGS) gene has recently been implicated in a novel congenital disorder of glycosylation resulting in autosomal recessive inherited glycosylphosphatidylinositol‐anchored protein (GPI‐AP) deficiency. Previous studies described seven patients with biallelic variants in the PIGS gene, of whom two presented with fetal akinesia and five with global developmental delay and epileptic developmental encephalopathy. We present the molecular and clinical characteristics of six additional individuals from five families with unreported variants in PIGS. All individuals presented with hypotonia, severe global developmental delay, microcephaly, intractable early infantile epilepsy, and structural brain abnormalities. Additional findings include vision impairment, hearing loss, renal malformation, and hypotonic facial appearances with minor dysmorphic features but without a distinctive facial gestalt. Four individuals died due to neurologic complications. GPI anchoring studies performed on one individual revealed a significant decrease in GPI‐APs. We confirm that biallelic variants in PIGS cause vitamin pyridoxine‐responsive epilepsy due to inherited GPI deficiency and expand the genotype and phenotype of PIGS‐related disorder. Further delineation of the molecular spectrum of PIGS‐related disorders would improve management, help develop treatments, and encourage the expansion of diagnostic genetic testing to include this gene as a potential cause of neurodevelopmental disorders and epilepsy.

Advances in the creation of animal models of paroxysmal nocturnal hemoglobinuria

ABSTRACT Paroxysmal nocturnal hemoglobinuria (PNH) is a disease caused by a phosphatidylinositol glycan anchor biosynthesis class A (PIG-A) mutation in hematopoietic stem cells. There are three theories about the possible mechanism of the pathogenesis of PNH: immune escape, anti-apoptotic mechanism, and secondary gene mutation. There has been little gain in the knowledge regarding its pathogenesis during the last decade owing to the lack of representative cell lines and animal models. There have been recent reports about the successful creation of PNH mouse and PNH rhesus macaque models. The detection of glycosylphosphatidylinositol-anchor protein (GPI-AP)-deficient cells and/or fluorescently labeled variant of aerolysin (FLAER) test, estimation of erythrocyte life span, and hemolysis-related experiments demonstrated that these animal models of PNH had GPI-AP-deficient blood cells with shortened lifespans and increased sensitivity to complement-activated hemolysis. However, there were no clinical manifestations such as hemolysis and thrombosis in these animal models. This suggested that the PIG-A mutation is one of the several conditions required for PNH, but it alone is not enough to cause PNH.

Deciphering the premature mortality in PIGA-CDG – An untold story

ObjectiveCongenital disorder of glycosylation (CDG) due to a defective phosphatidylinositol glycan anchor biosynthesis class A protein (PIGA) is a severe X-linked developmental and epileptic encephalopathy. Seizures are often treatment refractory, and patients have intellectual disability and global developmental delay. Previous reports have suggested that patients with PIGA-CDG have a high risk of premature mortality. This study aimed to evaluate the observed high mortality and the causes of death in PIGA-CDG patients. MethodsWe reviewed the literature and collected additional unpublished patients through an international network. ResultsIn total, we reviewed the data of 88 patients of whom 30 patients born alive were deceased, and the overall mortality before the age of 20 years was 30 % (26/88). Age at death ranged from 15 days to 48 years of life. The median age at death was two years and more than half of the patients deceased in early childhood. The PIGA-specific mortality rate/1000 person-years was 44.9/1000 person-years (95 %, CI 31.4–64.3). There were no cases of definite or probable sudden unexpected death in epilepsy (SUDEP) and half of the patients died due to respiratory failure (15/30, 50 %) or possible SUDEP (3/30, 10 %). Three patients (10 %) died from severe cardiomyopathy, liver failure and gastrointestinal bleeding, respectively. The cause of death was unclassified in nine patients (30 %). Autopsies were rarely performed and the true cause of death remains unknown for the majority of patients. SignificanceOur data indicate an increased risk of premature death in patients with PIGA-CDG when compared to most monogenic developmental and epileptic encephalopathies.

PIGH deficiency can be associated with severe neurodevelopmental and skeletal manifestations.

Phosphatidylinositol Glycan Anchor Biosynthesis class H (PIGH) is an essential player in the glycosylphosphatidylinositol (GPI) synthesis, an anchor for numerous cell membrane‐bound proteins. PIGH deficiency is a newly described and rare disorder associated with developmental delay, seizures and behavioral difficulties. Herein, we report three new unrelated families with two different bi‐allelic PIGH variants, including one new variant p.(Arg163Trp) which seems associated with a more severe phenotype. The common clinical features in all affected individuals are developmental delay/intellectual disability and hypotonia. Variable clinical features include seizures, autism spectrum disorder, apraxia, severe language delay, dysarthria, feeding difficulties, facial dysmorphisms, microcephaly, strabismus, and musculoskeletal anomalies. The two siblings homozygous for the p.(Arg163Trp) variant have severe symptoms including profound psychomotor retardation, intractable seizures, multiple bone fractures, scoliosis, loss of independent ambulation, and delayed myelination on brain MRI. Serum iron levels were significantly elevated in one individual. All tested individuals with PIGH deficiency had normal alkaline phosphatase and CD16, a GPI‐anchored protein (GPI‐AP), was found to be decreased by 60% on granulocytes from one individual. This study expands the PIGH deficiency phenotype range toward the severe end of the spectrum with the identification of a novel pathogenic variant.

Paroxysmal nocturnal haemoglobinuria (PNH): novel therapies for an ancient disease.

In the UK, early work on paroxysmal nocturnal haemoglobinuria (PNH) was conducted by John Dacie who, at the Hammersmith Hospital, first hypothesised that the PNH abnormality might arise through a somatic mutation; and who outlined with S.M. Lewis the relationship between PNH and aplastic anaemia. When the phosphatidylinositol glycan anchor biosynthesis Class A (PIGA) gene was identified by Taroh Kinoshita's group, jointly with him the Hammersmith group proved that PNH is caused in most patients by a single somatic mutation in the PIGA gene. At the same time, after Bruno Rotoli had spent a sabbatical at the Hammersmith, the 'immune escape model' for the pathogenesis of PNH was developed. Early this century, Peter Hillmen, formerly at the Hammersmith and now in Leeds, spearheaded the use of the complement-blocking (anti-C5) antibody eculizumab. This new medicine radically changed the management and the clinical course of patients with PNH. Recently a derivative of eculizumab with more favourable pharmacokinetics has been introduced. In view of the fact that these agents are associated with C3-dependent extravascular haemolysis, it is important that a number of inhibitors of the proximal complement pathway are now in the offing and may further improve the life of patients with PNH.

PIGU promotes hepatocellular carcinoma progression through activating NF-κB pathway and increasing immune escape

Hepatocellular carcinoma (HCC) is the sixth most common malignancy and has the third highest mortality rate among all tumors. Previous studies found that phosphatidylinositol glycan anchor biosynthesis class U (PIGU) was highly expressed in hepatocellular carcinoma (HCC), while the function of PIGU in HCC remains unknown. Here, we deeply investigated this issue. The expression levels of PIGU in HCC cells were measured by Western blotting. The functions of PIGU in HCC cells were assessed in vitro, followed by assessing the nuclear factor-kappa B (NF-κB) pathway-related protein levels. The xenograft mouse models were conducted to investigate the effects of PIGU in vivo. Moreover, the effects of PIGU downregulation on natural killer (NK)-92 cell-mediated cell killing were detected. The results showed that PIGU was highly expressed in HCC cells compared with normal liver cells. Functional studies showed that PIGU promoted viability, cell cycle progression, migration, and invasion and suppressed apoptosis in HCC cells. Mechanism studies indicated that PIGU silencing blocked the NF-κB pathway and the blockade of the NF-κB pathway reversed the effects of PIGU overexpression on HCC cell function, including cell viability, migration, invasion, and apoptosis. In vivo studies further verified the effects of PIGU on HCC cell function, and demonstrated that PIGU knockdown suppressed tumorigenesis. Additionally, we proved that PIGU downregulation significantly enhanced the sensitivity of HCC cells to NK-92 cell cytolysis. Collectively, PIGU may promote HCC progression through activating the NF-κB pathway and promoting immune escape, indicating that PIGU may serve as a promising therapeutic target for HCC treatment.

132P MALAT-1 ruling the miR-182/PIG-C/MSLN triad in triple negative breast cancer

In the last decade, cancer immune evasion has brought immunotherapy to the light of oncology and became a principle approach in cancer therapy. microRNA-34a and Long non-coding RNA MALAT1 are examples of a newly emerged group of non-Coding RNAs that have grabbed attention towards understanding epigenetic manipulation in many cancers among which is triple negative breast cancer (TNBC) that is characterized by strong heterogeneity and aggressiveness. Our previous data revealed that Mesothelin (MSLN) and phosphatidylinositol glycan anchor biosynthesis class C (PIG-C) are overexpressed in breast cancer cell lines and biopsies.

Radioiodine-Refractory Thyroid Cancer: Molecular Basis of Redifferentiation Therapies, Management, and Novel Therapies.

Recurrent, metastatic disease represents the most frequent cause of death for patients with thyroid cancer, and radioactive iodine (RAI) remains a mainstay of therapy for these patients. Unfortunately, many thyroid cancer patients have tumors that no longer trap iodine, and hence are refractory to RAI, heralding a poor prognosis. RAI-refractory (RAI-R) cancer cells result from the loss of thyroid differentiation features, such as iodide uptake and organification. This loss of differentiation features correlates with the degree of mitogen-activated protein kinase (MAPK) activation, which is higher in tumors with BRAF (B-Raf proto-oncogene) mutations than in those with RTK (receptor tyrosine kinase) or RAS (rat sarcoma) mutations. Hence, inhibition of the mitogen-activated protein kinase kinase-1 and -2 (MEK-1 and -2) downstream of RAF (rapidly accelerated fibrosarcoma) could sensitize RAI refractivity in thyroid cancer. However, a significant hurdle is the development of secondary tumor resistance (escape mechanisms) to these drugs through upregulation of tyrosine kinase receptors or another alternative signaling pathway. The sodium iodide symporter (NIS) is a plasma membrane glycoprotein, a member of solute carrier family 5A (SLC5A5), located on the basolateral surfaces of the thyroid follicular epithelial cells, which mediates active iodide transport into thyroid follicular cells. The mechanisms responsible for NIS loss of function in RAI-R thyroid cancer remains unclear. In a study of patients with recurrent thyroid cancer, expression levels of specific ribosomal machinery—namely PIGU (phosphatidylinositol glycan anchor biosynthesis class U), a subunit of the GPI (glycosylphosphatidylinositol transamidase complex—correlated with RAI avidity in radioiodine scanning, NIS levels, and biochemical response to RAI treatment. Here, we review the proposed mechanisms for RAI refractivity and the management of RAI-refractive metastatic, recurrent thyroid cancer. We also describe novel targeted systemic agents that are in use or under investigation for RAI-refractory disease, their mechanisms of action, and their adverse events.

Abnormal expression and mutation of the RBPJ gene may be involved in CD59- clonal proliferation in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal proliferative disease of hematopoietic stem cells. Various gene mutations, including the phosphatidylinositol glycan anchor biosynthesis class A (PIG-A) gene, may contribute to the proliferation of PNH clones. In order to explore the mechanism of PNH clone proliferation, a study was performed on 13 patients with PNH who underwent whole exome sequencing. The frequency of mutations in these patients was explored, and an additional 30 patients with PNH were selected for analysis of cluster of differentiation 59-negative (CD59−) cells. The mRNA expression of 13 genes, which were selected based on their high frequency in patients with PNH and the fact that they met four screening conditions, was determined in these CD59− cells. Cell proliferation, apoptosis and cell cycle were evaluated upon knocking down the recombinant signal binding protein of immunoglobulin κJ region (RBPJ) gene in 5 patients in vitro. The detection rate of PIG-A gene mutation was 61.54% (8/13), and additional mutations in somatic genes were detected, including RBPJ, zinc finger protein 717, polycomb repressive complex 2 subunit and tet methylcytosine dioxygenase. Upon screening according to the mutation frequency and expression level, the present study focused on the RBPJ gene. The expression level of RBPJ in CD59− cells was apparently higher than that in CD59+ cells and normal controls which was significantly correlated with clinical data. Furthermore, the expression of RBPJ in PNH primary cells could be effectively inhibited by small interfering RNA-RBPJ. Once the expression of RBPJ decreased remarkably, the apoptotic rate increased gradually, while cell proliferation activity decreased with transfection time and cells were blocked in G0/G1 phase. In conclusion, mutations and abnormal expression of the RBPJ gene may participate in the abnormal proliferation of PNH clones.