A superior heterologous prime-boost vaccination strategy against COVID-19: A bivalent vaccine based on yeast-derived RBD proteins followed by a heterologous vaccine.

Various vaccines have been challenged by SARS-CoV-2 variants. Here, we reported a yeast-derived recombinant bivalent vaccine (Bivalent wild-type [Wt]+De) based on the wt and Delta receptor-binding domain (RBD). Yeast derived RBD proteins based on the wt and Delta mutant were used as the prime vaccine. It was found that, in the presence of aluminium hydroxide (Alum) and unmethylated CpG-oligodeoxynucleotides (CpG) adjuvants, more cross-protective immunity against SARS-CoV-2 prototype and variants were elicited by bivalent vaccine than monovalent wtRBD or Delta RBD. Furthermore, a heterologous boosting strategy consisting of two doses of bivalent vaccines followed by one dose adenovirus vectored vaccine exhibited cross-neutralization capacity and specific T cell responses against Delta and Omicron (BA.1 and BA.4/5) variants in mice, superior to a homologous vaccination strategy. This study suggested that heterologous prime-boost vaccination with yeast-derived bivalent protein vaccine could be a potential approach to address the challenge of emerging variants.

Enhanced sensitivity of neutralizing antibody detection for different AAV serotypes using HeLa cells with overexpressed AAVR.

A cell-based transduction inhibition assay (TI) is widely used in clinical trials to detect neutralizing antibody (NAb) titers against recombinant adeno-associated virus (rAAV), one of the most important criteria to exclude patients in gene therapy. Different cell lines are used in cell-based TI because the rAAV transduction efficiencies vary largely among serotypes. A cell line suitable for TI for most serotypes is highly desirable, especially for those with very low transduction efficiencies in vitro such as rAAV8 and rAAV9. Herein, we report an AAVR-HeLa, a stable cell line with overexpressed AAVR, a newly identified receptor for rAAVs, was established for cell-based TIs. The AAVR expression level in AAVR-HeLa cells was approximately 10-fold higher than in HeLa cells, and was stably transfected after twenty three passages. For all AAV serotypes (AAV1-10), except for AAV4, the transduction efficiencies increased significantly in AAVR-HeLa cells. It was demonstrated that the AAVR enhancement of transduction efficiency was only for rAAV and not for lentiviral and adenoviral vectors. According to the minimal multiplicity of infection (MOIs) for the assay, the NAb detection sensitivity increased at least 10 and 20 fold for AAV8 and AAV9, respectively. The seroprevalence of NAbs were investigated at the 1:30 level as a cutoff value using AAVR-HeLa cells. It was shown that the seropositive rate for AAV2 was 87% in serum samples from 99 adults, followed by lower seropositive rates for AAV5 (7%), AAV8 (7%) and AAV9 (1%). Venn diagram analysis showed the presence of cross-reactivity of NAbs to two or three serotypes in 13 samples (13.1%). However, no patient was found to possess NAbs for all the four serotypes. These results demonstrated that the AAVR-HeLa cell line may be utilized to detect the NAbs through cell-based TI assays for most of AAV serotypes.

Centenarians Alleviate Inflammaging by Changing the Ratio and Secretory Phenotypes of T Helper 17 and Regulatory T Cells.

The immune system of centenarians remains active and young to prevent cancer and infections. Aging is associated with inflammaging, a persistent low-grade inflammatory state in which CD4+ T cells play a role. However, there are few studies that have been done on the CD4+ T cell subsets in centenarians. Herein, the changes in CD4+ T cell subsets were investigated in centenarians. It was found that with aging, the old adults had higher levels of proinflammatory cytokines and lower levels of anti-inflammatory cytokines in plasma. The levels of CRP, IL-12, TNF-α, IFN-γ, IL-6 and IL-10 were further increased in centenarians compared to old adults. While the levels of IL-17A, IL-1ß, IL-23 and TGF-ß in centenarians were closer to those in young adults. The total CD4+, CD8+, Th17 and Treg cells from peripheral blood mononuclear cells (PBMCs) were similar among the three groups. It was observed that the ratio of Th17/Treg cells was elevated in old adults compared to young adults. The ratio was not further elevated in centenarians but rather decreased. In addition, the ex vivo PBMCs differentiation assay showed that increased Th17 cells in centenarians tended to secrete fewer proinflammatory cytokines, while decreased Treg cells in centenarians were prone to secrete more anti-inflammatory cytokines. These observations suggested centenarians alleviated inflammaging by decreasing the ratio of Th17/Treg cells and changing them into anti-inflammatory secretory phenotypes, which provided a novel mechanism for anti-aging research.

Development of an Adeno-Associated Virus-Vectored SARS-CoV-2 Vaccine and Its Immunogenicity in Mice.

Owing to the outbreak of the novel coronavirus (SARS-CoV-2) worldwide at the end of 2019, the development of a SARS-CoV-2 vaccine became an urgent need. In this study, we developed a type 9 adeno-associated virus vectored vaccine candidate expressing a dimeric receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) and evaluated its immunogenicity in a murine model. The vaccine candidate, named AAV9-RBD virus, was constructed by inserting a signal peptide to the N-terminus of two copies of RBD, spaced by a linker, into the genome of a type 9 adeno-associated virus. In vitro assays showed that HeLa cells infected by the recombinant AAV virus expressed high levels of the recombinant RBD protein, mostly found in the cell culture supernatant. The recombinant AAV9-RBD virus was cultured and purified. The genome titer of the purified recombinant AAV9-RBD virus was determined to be 2.4 × 1013 genome copies/mL (GC/mL) by Q-PCR. Balb/c mice were immunized with the virus by intramuscular injection or nasal drip administration. Eight weeks after immunization, neutralizing antibodies against the new coronavirus pseudovirus were detected in the sera of all mice; the mean neutralizing antibody EC50 values were 517.7 ± 292.1 (n=10) and 682.8 ± 454.0 (n=10) in the intramuscular injection group and nasal drip group, respectively. The results of this study showed that the recombinant AAV9-RBD virus may be used for the development of a SARS-CoV-2 vaccine.

Optimization of miR-22 expression cassette for rAAV delivery on diabetes.

MicroRNA-22 (miR-22) was suggested to be important for type 2 diabetes but its functions for this disease remained unclear. Recombinant adeno-associated virus (rAAV)-mediated miR delivery is a powerful approach to study miR functions in vivo, however, the overexpression of miR-22 by rAAV remains challenging because it is one of the most abundant miRs in the liver. In this study, a series of expression cassettes were designed and compared. It was shown that different lengths of primary miR-22 were overexpressed in HEK293 and HeLa cells but the longer ones were more efficiently expressed. miR-22 may be placed in either introns or the 3' UTR of a transgene for efficient overexpression. RNA polymerase III or II promoters were successfully utilized for miR expression but the latter showed higher expression levels in cell lines. Specifically, miR-22 was expressed efficiently together with an EGFP gene. After screening, a liver-specific TTR promoter was chosen to overexpress miR-22 in diabetic mice fed a high-fat diet. It was shown that miR-22 was overexpressed 2-3 folds which improved the insulin sensitivity significantly. The approach utilized in this study to optimize miR overexpression is a powerful tool for the creation of efficient rAAV vectors for the other miRs.

Deleterious Variants in ABCC12 are Detected in Idiopathic Chronic Cholestasis and Cause Intrahepatic Bile Duct Loss in Model Organisms.

BACKGROUND & AIMS: The etiology of cholestasis remains unknown in many children. We surveyed the genome of children with chronic cholestasis for variants in genes not previously associated with liver disease and validated their biological relevance in zebrafish and murine models. METHOD: Whole-exome (n = 4) and candidate gene sequencing (n = 89) was completed on 93 children with cholestasis and normal serum γ-glutamyl transferase (GGT) levels without pathogenic variants in genes known to cause low GGT cholestasis such as ABCB11 or ATP8B1. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing was used to induce frameshift pathogenic variants in the candidate gene in zebrafish and mice. RESULTS: In a 1-year-old female patient with normal GGT cholestasis and bile duct paucity, we identified a homozygous truncating pathogenic variant (c.198delA, p.Gly67Alafs∗6) in the ABCC12 gene (NM_033226). Five additional rare ABCC12 variants, including a pathogenic one, were detected in our cohort. ABCC12 encodes multidrug resistance-associated protein 9 (MRP9) that belongs to the adenosine 5'-triphosphate-binding cassette transporter C family with unknown function and no previous implication in liver disease. Immunohistochemistry and Western blotting revealed conserved MRP9 protein expression in the bile ducts in human, mouse, and zebrafish. Zebrafish abcc12-null mutants were prone to cholangiocyte apoptosis, which caused progressive bile duct loss during the juvenile stage. MRP9-deficient mice had fewer well-formed interlobular bile ducts and higher serum alkaline phosphatase levels compared with wild-type mice. They exhibited aggravated cholangiocyte apoptosis, hyperbilirubinemia, and liver fibrosis upon cholic acid challenge. CONCLUSIONS: Our work connects MRP9 with bile duct homeostasis and cholestatic liver disease for the first time. It identifies a potential therapeutic target to attenuate bile acid-induced cholangiocyte injury.

Identification of glucocorticoid receptor in Drosophila melanogaster.

BACKGROUND: Vertebrate glucocorticoid receptor (GR) is an evolutionary-conserved cortisol-regulated nuclear receptor that controls key metabolic and developmental pathways. Upon binding to cortisol, GR acts as an immunosuppressive transcription factor. Drosophila melanogaster, a model organism to study innate immunity, can also be immunosuppressed by glucocorticoids. However, while the genome of fruit fly harbors 18 nuclear receptor genes, the functional homolog of vertebrate GR has not been identified. RESULTS: In this study, we demonstrated that while D. melanogaster is susceptible to Saccharomyces cerevisiae oral infection, the oral exposure to cortisol analogs, cortisone acetate or estrogen, increases fly sensitivity to yeast challenge. To understand the mechanism of this steroid-induced immunosuppression, we identified the closest genetic GR homolog as D. melanogaster Estrogen Related Receptor (ERR) gene. We discovered that Drosophila ERR is necessary for cortisone acetate- and estrogen-mediated increase in sensitivity to fungal infection: while ERR mutant flies are as sensitive to the fungal challenge as the wildtype flies, the yeast-sensitivity of ERR mutants is not increased by these steroids. Interestingly, the fungal cortisone analog, ergosterol, did not increase the susceptibility of Drosophila to yeast infection. The immunosuppressive effect of steroids on the sensitivity of flies to fungi is evolutionary conserved in insects, as we show that estrogen significantly increases the yeast-sensitivity of Culex quinquefasciatus mosquitoes, whose genome contains a close ortholog of the fly ERR gene. CONCLUSIONS: This study identifies a D. melanogaster gene that structurally resembles vertebrate GR and is functionally necessary for the steroid-mediated immunosuppression to fungal infections.

PAX1 is essential for development and function of the human thymus.

We investigated the molecular and cellular basis of severe combined immunodeficiency (SCID) in six patients with otofaciocervical syndrome type 2 who failed to attain T cell reconstitution after allogeneic hematopoietic stem cell transplantation, despite successful engraftment in three of them. We identified rare biallelic PAX1 rare variants in all patients. We demonstrated that these mutant PAX1 proteins have an altered conformation and flexibility of the paired box domain and reduced transcriptional activity. We generated patient-derived induced pluripotent stem cells and differentiated them into thymic epithelial progenitor cells and found that they have an altered transcriptional profile, including for genes involved in the development of the thymus and other tissues derived from pharyngeal pouches. These results identify biallelic, loss-of-function PAX1 mutations as the cause of a syndromic form of SCID due to altered thymus development.

Genetic variants in acute, acute recurrent and chronic pancreatitis affect the progression of disease in children.

BACKGROUND/OBJECTIVES: Acute pancreatitis (AP) is emerging in pediatrics. A subset of children with AP progresses to acute recurrent pancreatitis (ARP) and chronic pancreatitis (CP). The role of extensive gene testing in the progression has not been investigated previously. We have followed children enrolled in the registry and at our center for progression to ARP and CP after the first attack. METHODS: This study utilizes an extensive gene sequencing panel as a platform to evaluate the role of genetics in first attack AP, and the progression over time, from first attack to ARP and CP in children. RESULTS: Genes, with corresponding variants were involved in the 3 groups studied: AP, ARP and CP. We have shown that the presence of gene variants from the eight tested genes is enriched in the CP group compared to the AP and ARP groups. The presence of more than one gene was associated with CP (p = 0.01). SPINK1 mutation(s) was significantly associated with faster progression to ARP, (p = 0.04). Having a variant from CFTR, SPINK1 or PRSS1, was associated with the faster progression from AP to CP over time (p < 0.05). CONCLUSIONS: This study shows that genetics have a significant role in progression to ARP and CP from the first attack of pancreatitis.

Genetic and Transcriptomic Variation Linked to Neutrophil Granulocyte-Macrophage Colony-Stimulating Factor Signaling in Pediatric Crohn's Disease.

BACKGROUND: Granulocyte-macrophage colony-stimulating factor auto-antibodies (GMAbs) suppress neutrophil-extrinsic GM-CSF signaling and increase risk for stricturing behavior in Crohn's disease (CD). We aimed to define clinical, genomic, and functional associations with neutrophil-intrinsic GM-CSF signaling. METHODS: Missense mutations in CSF2RA, CSF2RB, JAK2, STAT5A, and STAT5B were identified using whole-exome sequencing in 543 pediatric inflammatory bowel disease (IBD) patients. Neutrophil-intrinsic GM-CSF signaling was defined using the GM-CSF-induced STAT5 stimulation index (GMSI) in 180 pediatric IBD patients and 26 non-IBD controls. Reduced GM-CSF signaling (GMSI-Lo) was defined as the 20th percentile within the control group. Variation in neutrophil phospho-protein abundance, bacterial killing, and the global pattern of gene expression with the GMSI was determined. RESULTS: We validated 18 potentially damaging missense mutations in CSF2RA and CSF2RB. CSF2RA A17G carriage increased from 10% in those with intact neutrophil GMSI to 32% in those with low GMSI (P = 0.02). The frequency of reduced Staphylococcus aureus killing increased from 17% in those with intact neutrophil GMSI to 35% in GMSI-Lo neutrophils (P = 0.043). Crohn's disease neutrophils with low GMSI exhibited specific alterations in phospho-protein networks and genes regulating cytokine production, wound healing, and cell survival and proliferation. Stricturing behavior increased from 7% in patients with both low GMAb and intact GMSI to 64% in patients with both elevated GMAb and low GMSI (P < 0.0001). CONCLUSIONS: Low/normal neutrophil-intrinsic GM-CSF signaling is associated with CSF2RA missense mutations, alterations in gene expression networks, and higher rates of disease complications in pediatric CD.

Prevalence of abnormal glucose metabolism in pediatric acute, acute recurrent and chronic pancreatitis.

Type 3C Diabetes, or diseases of the exocrine pancreas has been reported to occur in approximately 30% of adult patient with pancreatitis. The incidence of glucose abnormalities or risk factors that may predict the development of abnormal glucose in the pediatric pancreatitis population is not known. We performed a retrospective chart review from 1998-2016 for patients who carry the diagnosis of acute pancreatitis (AP), acute recurrent pancreatitis (ARP), and chronic pancreatitis (CP). We extracted glucose values, HbA1c%, and data from oral glucose tolerance and mixed meal testing with timing in relation to pancreatic exacerbations. Patient characteristic data such as age, gender, body proportions, family history of pancreatitis, exocrine function and genetic mutations were also assessed. Abnormal glucose was based on definitions put forth by the American Diabetes Society for pre-diabetes and diabetes. Fifty-two patients had AP and met criteria. Of those, 15 (29%) had glucose testing on or after the first attack, 21 (40%) were tested on or after the second attack (in ARP patients) and 16 (31%) were tested after a diagnosis of CP. Of the patients tested for glucose abnormalities, 25% (13/52) had abnormal glucose testing (testing indicating pre-DM or DM as defined by ADA guidelines. A significantly higher proportion of the abnormal glucose testing was seen in patients (85%, 11/13) with a BMI at or greater than the 85th percentile compared to the normal glucose patients (28%, 11/39) (p = 0.0007). A significantly higher proportion of the abnormal glucose patients (77%, 10/13) had SAP during the prior AP episode to testing compared to the 10% (4/39) of the normal glucose patients (p<0.0001). Older age at DM testing was associated with a higher prevalence of abnormal glucose testing (p = 0.04). In our patient population, a higher proportion of glucose abnormalities were after the second episode of pancreatitis, however 62% (8/13) with abnormalities was their first time tested. We identified obesity and having severe acute pancreatitis (SAP) during the prior AP episode to testing could be associated with abnormal glucose. We propose that systematic screening for abnormal glucose after the first episode of acute pancreatitis in order to better establish the timing of diabetes progression.

Zebrafish abcb11b mutant reveals strategies to restore bile excretion impaired by bile salt export pump deficiency.

Bile salt export pump (BSEP) adenosine triphosphate-binding cassette B11 (ABCB11) is a liver-specific ABC transporter that mediates canalicular bile salt excretion from hepatocytes. Human mutations in ABCB11 cause progressive familial intrahepatic cholestasis type 2. Although over 150 ABCB11 variants have been reported, our understanding of their biological consequences is limited by the lack of an experimental model that recapitulates the patient phenotypes. We applied CRISPR/Cas9-based genome editing technology to knock out abcb11b, the ortholog of human ABCB11, in zebrafish and found that these mutants died prematurely. Histological and ultrastructural analyses showed that abcb11b mutant zebrafish exhibited hepatocyte injury similar to that seen in patients with progressive familial intrahepatic cholestasis type 2. Hepatocytes of mutant zebrafish failed to excrete the fluorescently tagged bile acid that is a substrate of human BSEP. Multidrug resistance protein 1, which is thought to play a compensatory role in Abcb11 knockout mice, was mislocalized to the hepatocyte cytoplasm in abcb11b mutant zebrafish and in a patient lacking BSEP protein due to nonsense mutations in ABCB11. We discovered that BSEP deficiency induced autophagy in both human and zebrafish hepatocytes. Treatment with rapamycin restored bile acid excretion, attenuated hepatocyte damage, and extended the life span of abcb11b mutant zebrafish, correlating with the recovery of canalicular multidrug resistance protein 1 localization. CONCLUSIONS: Collectively, these data suggest a model that rapamycin rescues BSEP-deficient phenotypes by prompting alternative transporters to excrete bile salts; multidrug resistance protein 1 is a candidate for such an alternative transporter. (Hepatology 2018;67:1531-1545).

High-Density Recombinant Adeno-Associated Viral Particles are Competent Vectors for In Vivo Transduction.

Recombinant adeno-associated viral (rAAV) vectors have recently achieved clinical successes in human gene therapy. However, the commonly observed, heavier particles found in rAAV preparations have traditionally been ignored due to their reported low in vitro transduction efficiency. In this study, the biological properties of regular and high-density rAAV serotype 8 vectors, rAAVRD and rAAVHD, were systemically compared. Results demonstrated that both rAAVRD and rAAVHD exhibited similar DNA packaging profiles, while rAAVHD capsids contained fewer VP1 and VP2 proteins, indicating that the rAAVHD particles contained a higher DNA/protein ratio than that of rAAVRD particles. Dynamic light scattering and transmission electron microscopy data revealed that the diameter of rAAVHD was smaller than that of rAAVRD. In vitro, rAAVHD was two- to fourfold less efficient in transduction compared with rAAVRD. However, the transduction performance of rAAVHD and rAAVRD was similar in vivo. No significant difference in neutralizing antibody formation against rAAVRD and rAAVHD was observed, suggesting that the surface epitopes of rAAVRD and rAAVHD are congruent. In summary, the results of this study demonstrate that rAAVRD and rAAVHD are equally competent for in vivo transduction, despite their difference in vitro. Therefore, the use of rAAVHD vectors in human gene therapy should be further evaluated.

Genophenotypic Analysis of Pediatric Patients With Acute Recurrent and Chronic Pancreatitis.

OBJECTIVES: The aim of this study was to determine if comprehensive genetic testing was useful to identify genetic variants that discriminate chronic pancreatitis (CP) from acute recurrent pancreatitis (ARP) in a pediatric population. METHODS: We conducted a retrospective review of 50 patients enrolled in our institutional pancreatitis registry between April 2013 and January 2015. Genetic analysis of PRSS1, CFTR, SPINK1, and CTRC classified variants as mutations or variants of unknown clinical significance and the minor allele frequency of variants in our cohort was obtained. RESULTS: Genetic testing was obtained in 16/16 (100%) of CP and 29/34 (85%) of ARP patients. A total of 39 genetic variants were found in 27 (60%) of 45 subjects tested with 5 (11%) subjects having 2 different genes affected. Variant frequency was greatest in patients for CFTR (17/45, 38%) followed by SPINK1 (11/44, 25%), CTRC (2/27, 7%), and PRSS1 (2/44, 4%). CFTR variants were more likely in those with CP compared to ARP (63% and 24%, P = 0.01). CONCLUSIONS: This study is the first to find a higher rate of CFTR mutations in CP versus ARP groups using comprehensive genetic testing in a pediatric population.

Potential role of exosome-associated microRNA panels and in vivo environment to predict drug resistance for patients with multiple myeloma.

Multiple myeloma (MM) is the second most common hematologic neoplasms and an appropriate in vivo environment for myeloma cells has potential implications for initiation, progression, and metastasis of MM. Exosomes, entities carrying microRNAs (miRNAs) to target locations, participate in the cross-talk between myeloma cells and nonmalignant components of the in vivo environment. This study disclosed the emerging roles of circulating exosome-associated miRNAs in drug resistance (DR) of MM. To this end, the medical records of consecutively hospitalized MM patients, who received novel agents-based therapies, were analyzed. Then, an optimized procedure was established for exosome isolation and exosomal RNA analysis. The exosome-associated miRNA expression patterns for predicting bortezomib (Bz) resistance of MM were further examined using a microarray. In total, 204 patients were enrolled with DR rates of 36.5%, 73.1% and 81.8% in the bortezomib (Bz), thalidomide and lenalidomide containing groups. The serum total light chain ratio ≥ 100, CRP ≥ 20 mg/L, and the second-line usage increased risks of acquired Bz-resistance. Among 68 cases having genetic tests, a high risk factor for predicting de novo DR was 1q21 amplification, which also correlated with lower levels of cholesterol and LDL-C. Moreover, nano-sized exosomes were isolated with significantly increasing internal RNAs and down-regulation of exosomal miR-16-5p, miR-15a-5p and miR-20a-5p, miR-17-5p was revealed in the patients resistant to Bz. The routine workup of MM hardly suggested a value for DR prediction. The circulating exosomes carrying miRNAs provided a window that permits a better understanding of the in vivo intercellular crosstalk in MM patients.

Transfer of microRNAs by extracellular membrane microvesicles: a nascent crosstalk model in tumor pathogenesis, especially tumor cell-microenvironment interactions.

Anticancer treatments aiming at killing malignant cells have been applied for decades but have been unsuccessful at curing the disease. The modern concept of tumor microenvironment, especially angiogenesis, suggests that the tumor is not only composed of malignant cells, but also consists of other groups of cells that work together. Recently, genetic message transfer has been revealed between tumor cells and their microenvironment. The latest cell-derived vector, extracellular membrane microvesicles (EMVs), has been found to provide membrane protection and allowed to deliver genetic information beyond the cells. Additionally, EMV-associated microRNAs are involved in a variety of cellular pathways for tumor initiation and progression. Previous published reviews have focused on miRNA that included EMVs as a sensitive marker for tumor monitoring in clinical applications that are based on the alteration of their expression levels in conjunction with disease occurrence and progression. From the aspect of cellular crosstalk, this article will review the role of EMV-mediated microRNA transfer in tumor pathogenesis, including tumor treatment obstacles, history and features, and current research in inflammatory/immune pathologies, as well as in solid tumors and hematological malignancies. This nascent crosstalk model will provide a novel insight into complementing the classic mechanisms of intercellular communication and contribute to the potential therapeutic strategy via small RNA molecule-carrying EMVs for multimodality treatment of cancer.

Synergistic defects of different molecules in the cytotoxic pathway lead to clinical familial hemophagocytic lymphohistiocytosis.

Several molecules (LYST, AP3, RAB27A, STX11, STXBP2, MUNC13-4, and PRF1) have been associated with the function of cytotoxic lymphocytes. Biallelic defects in all of these molecules have been associated with familial hemophagocytic lymphohistiocytosis (FHL). We retrospectively reviewed the genetic and immunology test results from 2701 patients with a clinically suspected diagnosis of hemophagocytic lymphohistiocytosis and found 28 patients with single heterozygous mutations in 2 FHL-associated genes. Of these patients, 21 had mutations within PRF1 and a degranulation gene, and 7 were found to have mutations within 2 genes involved in the degranulation pathway. In patients with combination defects involving 2 genes in the degranulation pathway, CD107a degranulation was decreased, comparable to patients with biallelic mutations in one of the genes in the degranulation pathway. This suggests a potential digenic mode of inheritance of FHL as a result of a synergistic function effect within genes involved in cytotoxic lymphocyte degranulation.

The 253-kb inversion and deep intronic mutations in UNC13D are present in North American patients with familial hemophagocytic lymphohistiocytosis 3.

BACKGROUND: The mutations in UNC13D are responsible for familial hemophagocytic lymphohistiocytosis (FHL) type 3. A 253-kb inversion and two deep intronic mutations, c.118-308C > T and c.118-307G > A, in UNC13D were recently reported in European and Asian FHL3 patients. We sought to determine the prevalence of these three non-coding mutations in North American FHL patients and evaluate the significance of examining these new mutations in genetic testing. PROCEDURE: We performed DNA sequencing of UNC13D and targeted analysis of these three mutations in 1,709 North American patients with a suspected clinical diagnosis of hemophagocytic lymphohistiocytosis (HLH). RESULTS: The 253-kb inversion, intronic mutations c.118-308C > T and c.118-307G > A were found in 11, 15, and 4 patients, respectively, in which the genetic basis (bi-allelic mutations) explained 25 additional patients. Taken together with previously diagnosed FHL3 patients in our HLH patient registry, these three non-coding mutations were found in 31.6% (25/79) of the FHL3 patients. The 253-kb inversion, c.118-308C > T and c.118-307G > A accounted for 7.0%, 8.9%, and 1.3% of mutant alleles, respectively. Significantly, eight novel mutations in UNC13D are being reported in this study. To further evaluate the expression level of the newly reported intronic mutation c.118-307G > A, reverse transcription PCR and Western blot analysis revealed a significant reduction of both RNA and protein levels suggesting that the c.118-307G > A mutation affects transcription. CONCLUSIONS: These specified non-coding mutations were found in a significant number of North American patients and inclusion of them in mutation analysis will improve the molecular diagnosis of FHL3.