Reduced myeloid commitment and increased uptake by macrophages of stem cell-derived HPS2 neutrophils.

Hermansky-Pudlak syndrome type 2 (HPS2) is a rare autosomal recessive disorder, caused by mutations in the AP3B1 gene, encoding the ß3A subunit of the adapter protein complex 3. This results in mis-sorting of proteins within the cell. A clinical feature of HPS2 is severe neutropenia. Current HPS2 animal models do not recapitulate the human disease. Hence, we used induced pluripotent stem cells (iPSCs) of an HPS2 patient to study granulopoiesis. Development into CD15POS cells was reduced, but HPS2-derived CD15POS cells differentiated into segmented CD11b+CD16hi neutrophils. These HPS2 neutrophils phenocopied their circulating counterparts showing increased CD63 expression, impaired degranulation capacity, and intact NADPH oxidase activity. Most noticeable was the decrease in neutrophil yield during the final days of HPS2 iPSC cultures. Although neutrophil viability was normal, CD15NEG macrophages were readily phagocytosing neutrophils, contributing to the limited neutrophil output in HPS2. In this iPSC model, HPS2 neutrophil development is affected by a slower rate of development and by macrophage-mediated clearance during neutrophil maturation.

Transient anti-cytokine autoantibodies superimpose the hyperinflammatory response in Kawasaki disease and multisystem inflammatory syndrome in children: a comparative cohort study on correlates of disease.

BACKGROUND: Children with SARS-CoV-2 related Multisystem Inflammatory Syndrome in Children (MIS-C) often present with clinical features that resemble Kawasaki disease (KD). Disease severity in adult COVID-19 is associated to the presence of anti-cytokine autoantibodies (ACAAs) against type I interferons. Similarly, ACAAs may be implicated in KD and MIS-C. Therefore, we explored the immunological response, presence of ACAAs and disease correlates in both disorders. METHODS: Eighteen inflammatory plasma protein levels and seven ACAAs were measured in KD (n = 216) and MIS-C (n = 56) longitudinally by Luminex and/or ELISA. Levels (up to 1 year post-onset) of these proteins were related to clinical data and compared with healthy paediatric controls. FINDINGS: ACAAs were found in both patient groups. The presence of ACAAs lagged behind the inflammatory plasma proteins and peaked in the subacute phase. ACAAs were mostly directed against IFN-γ (>80%) and were partially neutralising at best. KD presented with a higher variety of ACAAs than MIS-C. Increased levels of anti-IL-17A (P = 0·02) and anti-IL-22 (P = 0·01) were inversely associated with ICU admission in MIS-C. Except for CXCL10 in MIS-C (P = 0·002), inflammatory plasma proteins were elevated in both KD and MIS-C. Endothelial angiopoietin-2 levels were associated with coronary artery aneurysms in KD (P = 0·02); and sCD25 (P = 0·009), angiopoietin-2 (P = 0·001), soluble IL-33-receptor (ST2, P = 0·01) and CXCL10 (P = 0·02) with ICU admission in MIS-C. INTERPRETATION: Markers of endothelial activation (E-selectin, angiopoietin-2), and innate and adaptive immune responses (macrophages [CD163, G-CSF], neutrophils [lipocalin-2], and T cells [IFN-γ, CXCL10, IL-6, IL-17]), are upregulated in KD and MIS-C. ACAAs were detected in both diseases and, although only partly neutralising, their transient presence and increased levels in non-ICU patients may suggest a dampening role on inflammation. FUNDING: The Kawasaki study is funded by the Dutch foundation Fonds Kind & Handicap and an anonymous donor. The sponsors had no role in the study design, analysis, or decision for publication.

Measuring Cannulation Skills for Hemodialysis: Objective Versus Subjective Assessment.

Lack of cannulation skill during hemodialysis treatments results in poor clinical outcomes due to infiltration and other cannulation-related trauma. Unfortunately, training of patient care technicians and nurses, specifically on the "technical" aspects of cannulation, has traditionally not received much attention. Simulators have been successfully deployed in many medical specialties for assessment and training of clinical skills. However, simulators have not been as widely used in nursing, especially in the context of training clinical personnel in the dialysis unit. We designed a state-of-the-art simulator for quantifying skill for hemodialysis cannulation. In this study, 52 nurses and patient care technicians with varying levels of clinical experience performed 16 cannulations on the simulator with different fistula properties. We formulated a composite metric for objectively measuring overall success of cannulation and compared this metric with subjective assessment by experts. In addition, we examined if years of clinical experience correlated with objective and subjective scores for cannulation skill. Results indicated that, while subjective and objective metrics generally correlated with each other, the objective metric was more precise and better suited for quantifying cannulation skill. Further, the simulator-based objective metric provides several advantages over subjective ratings, including providing fine-grained assessment of skill, consistency in measurement unaffected by subjective biases, and basing assessment on a more complete evaluation of performance. Years of clinical experience, however, demonstrated little correlation with either method of skill assessment. The methods presented for cannulation skill assessment in this study, if widely applied, could result in improved cannulation skill among our PCTs and nurses, which could positively impact patient outcomes in a tangible way.

Generation and characterization of a human iPSC line SANi008-A from a Chédiak-Higashi Syndrome patient.

Induced pluripotent stem cells (iPSCs) were generated from erythroblasts (EBLs) obtained from a patient diagnosed with Chédiak-Higashi Syndrome (CHS), caused by mutations in LYST (c.4322_4325delAGAG and c.10127A>G). EBLs were reprogrammed with CytoTune-iPS 2.0 Sendai Reprogramming Kit, where the generated iPSCs showed normal karyotype, expression of pluripotency associated markers and in vitro spontaneous differentiation towards the three germ layers. The generated iPSCs can be used to study CHS pathophysiology and the role of LYST in different cell types.

Generation and characterization of a human iPSC line SANi007-A from a patient with a heterozygous dominant mutation in ELANE.

Induced pluripotent stem cell (iPSC) line was generated from erythroblasts (EBLs) derived from a patient diagnosed with severe congenital neutropenia, caused by mutations in ELANE (c.614delG). Transgene-free iPSC line was generated using Sendai virus reprogramming. The iPSC line showed normal karyotype, expressed pluripotency associated genes and was capable of in vitro spontaneous differentiation towards the three germ layers. The generated iPSC line can be used to study severe congenital neutropenia and the role of neutrophil elastase protein.

Generation and characterization of a human iPSC line SANi006-A from a Gray Platelet Syndrome patient.

Induced pluripotent stem cells (iPSCs) were generated from erythroblasts (EBLs) obtained from a patient diagnosed with Gray Platelet Syndrome (GPS), caused by compound heterozygous NBEAL2 mutations (c.6568delT and c.7937T>C). GPS is an autosomal recessive bleeding disorder characterized by a lack of α-granules in platelets and progressive myelofibrosis. EBLs were reprogrammed with CytoTune-iPS 2.0 Sendai Reprogramming Kit, where the generated iPSCs showed normal karyotype, expression of pluripotency associated markers and in vitro spontaneous differentiation towards the three germ layers. The generated iPSCs can be used to study GPS pathophysiology and the basic functions of NBEAL2 protein in different cell types.

Generation and characterization of a control and patient-derived human iPSC line containing the Hermansky Pudlak type 2 (HPS2) associated heterozygous compound mutation in AP3B1.

Induced pluripotent stem cells (iPSCs) were generated from blood outgrowth endothelial cells (BOECs) obtained from a healthy donor and from a patient diagnosed with Hermansky Pudlak Syndrome type 2 (HPS2), caused by compound heterozygous AP3B1 mutations (c.177delA and c.1839-1842delTAGA). BOECs were reprogrammed with a hOKSM self-silencing polycistronic lentiviral vector, where the generated iPSCs showed normal karyotype, expression of pluripotency associated markers and in vitro spontaneous differentiation towards the three germ layers. The generated iPSCs can be used to study HPS2 pathophysiology and the basic functions of AP3B1 protein in different cell types.

SIRPα on Mouse B1 Cells Restricts Lymphoid Tissue Migration and Natural Antibody Production.

The inhibitory immunoreceptor SIRPα is expressed on myeloid and neuronal cells and interacts with the broadly expressed CD47. CD47-SIRPα interactions form an innate immune checkpoint and its targeting has shown promising results in cancer patients. Here, we report expression of SIRPα on B1 lymphocytes, a subpopulation of murine B cells responsible for the production of natural antibodies. Mice defective in SIRPα signaling (SIRPαΔCYT mice) displayed an enhanced CD11b/CD18 integrin-dependent B1 cell migration from the peritoneal cavity to the spleen, local B1 cell accumulation, and enhanced circulating natural antibody levels, which was further amplified upon immunization with T-independent type 2 antigen. As natural antibodies are atheroprotective, we investigated the involvement of SIRPα signaling in atherosclerosis development. Bone marrow (SIRPαΔCYT>LDLR-/-) chimaeric mice developed reduced atherosclerosis accompanied by increased natural antibody production. Collectively, our data identify SIRPα as a unique B1 cell inhibitory receptor acting to control B1 cell migration, and imply SIRPα as a potential therapeutic target in atherosclerosis.

The association and functional relevance of genetic variation in low-to-medium-affinity Fc-gamma receptors with clinical platelet transfusion refractoriness.

BACKGROUND: Inadequate responses to platelet transfusions (i.e., platelet transfusion refractoriness [PLT refractoriness]) are a serious problem. Multiple factors contribute to low yields upon platelet transfusion, among which are platelet-reactive allo-antibodies. Platelet-reactive allo-antibodies occur in up to 30% of patients receiving multiple transfusions, and presumably lead to rapid destruction of the transfused platelets via receptors for IgG, the Fc-gamma receptors (FcγRs). Genetic variation in FcγRs is associated with susceptibility to immune thrombocytopenia, in which autoantibodies against platelets cause thrombocytopenia. OBJECTIVES: We hypothesized that genetic variation in FcγRs may also influence PLT refractoriness in allo-immunized patients and could help in identifying the patients at risk. PATIENTS/METHODS: Patients with severe PLT refractoriness for whom diagnostic testing for allo-immunization was requested in the period of 2005 to 2013 were retrospectively included. A case-control study was performed comparing patients in whom platelet-reactive antibodies were detected (n = 181) with ethnically matched healthy controls (n = 180) to determine differences in all known functional copy number variations and single nucleotide polymorphisms in FcγRs. RESULTS AND CONCLUSIONS: None of the tested FcγR genetic variations seemed associated with the development of severe PLT refractoriness. In contrast to observations in immune thrombocytopenia, genetic variation in FcγRs does not seem to influence the chance to develop PLT refractoriness. Our results do not support determination of FcγR genetic background as a means to identify patients most at risk for PLT refractoriness.

MKL1 deficiency results in a severe neutrophil motility defect due to impaired actin polymerization.

Megakaryoblastic leukemia 1 (MKL1) promotes the regulation of essential cell processes, including actin cytoskeletal dynamics, by coactivating serum response factor. Recently, the first human with MKL1 deficiency, leading to a novel primary immunodeficiency, was identified. We report a second family with 2 siblings with a homozygous frameshift mutation in MKL1. The index case died as an infant from progressive and severe pneumonia caused by Pseudomonas aeruginosa and poor wound healing. The younger sibling was preemptively transplanted shortly after birth. The immunodeficiency was marked by a pronounced actin polymerization defect and a strongly reduced motility and chemotactic response by MKL1-deficient neutrophils. In addition to the lack of MKL1, subsequent proteomic and transcriptomic analyses of patient neutrophils revealed actin and several actin-related proteins to be downregulated, confirming a role for MKL1 as a transcriptional coregulator. Degranulation was enhanced upon suboptimal neutrophil activation, whereas production of reactive oxygen species was normal. Neutrophil adhesion was intact but without proper spreading. The latter could explain the observed failure in firm adherence and transendothelial migration under flow conditions. No apparent defect in phagocytosis or bacterial killing was found. Also, monocyte-derived macrophages showed intact phagocytosis, and lymphocyte counts and proliferative capacity were normal. Nonhematopoietic primary fibroblasts demonstrated defective differentiation into myofibroblasts but normal migration and F-actin content, most likely as a result of compensatory mechanisms of MKL2, which is not expressed in neutrophils. Our findings extend current insight into the severe immune dysfunction in MKL1 deficiency, with cytoskeletal dysfunction and defective extravasation of neutrophils as the most prominent features.

A Homozygous Mutation on the HBA1 Gene Coding for Hb Charlieu (HBA1: c.320T>C) Together with ß-Thalassemia Trait Results in Severe Hemolytic Anemia.

A 4-year-old boy, a ß-thalassemia (ß-thal) carrier, with an unexplained severe chronic microcytic anemia was referred to us. Sequencing of the α-globin genes revealed a Hb Charlieu [α106(G13)Leu→Pro, HBA1: c.320T>C, p.Leu107Pro] mutation present on both HBA1 genes. Quantitative polymerase chain reaction (qPCR) confirmed αCharlieu mRNA in the proband and his parents, showing that the mutation does not affect mRNA stability. However, we were unable to detect the Hb Charlieu protein by capillary electrophoresis (CE), reverse phase electrophoresis, cation exchange electrophoresis or isoelectric focusing. Mass spectrometry (MS) allowed us to confirm the presence of the Hb Charlieu peptide in erythrocyte progenitors. These findings suggest that the mutation affects the stability of αCharlieu. As hemoglobin (Hb) heat stability tests showed no abnormalities in erythrocytes, we speculated that αCharlieu is already degraded during red blood cell (RBC) development. The clinical severity in the proband and the presence of new methylene blue-stained aggregates in his reticulocytes indicates that incorporation of αCharlieu destabilizes Hb. This, combined with an excess of unstable free α-globins as the result of ß-thal minor, results in severely impaired erythropoiesis and, as a consequence, severe and chronic microcytic anemia in the proband.

Neutrophils acquire antigen-presenting cell features after phagocytosis of IgG-opsonized erythrocytes.

Neutrophils are particularly well known for their antimicrobial function. Although historically they are regarded as strictly a phagocyte of the innate immune system, over time it has become clear that neutrophils are versatile cells with numerous functions including innate and adaptive immune regulation. We have previously described a role for human neutrophils in antibody-mediated red blood cell (RBC) clearance. Under homeostatic conditions, neutrophils do not take up RBCs. Yet, when RBCs are immunoglobulin G (IgG) opsonized, which can occur in alloimmunization or autoimmunization reactions, neutrophils can effectively phagocytose RBCs. In the present study, we show that human neutrophils acquire an antigen-presenting cell (APC) phenotype following RBC phagocytosis. Subsequent to RBC phagocytosis, neutrophils expressed major histocompatibility complex class II (MHC-II) and costimulatory molecules such as CD40 and CD80. Moreover, in classical APCs, the respiratory burst is known to regulate antigen presentation. We found that the respiratory burst in neutrophils is reduced after IgG-mediated RBC phagocytosis. Additionally, following RBC phagocytosis, neutrophils were demonstrated to elicit an antigen-specific T-cell response, using tetanus toxoid (TT) as an antigen to elicit an autologous TT-specific CD4+ T-cell response. Lastly, although the "don't eat me" signal CD47 is known to have a powerful restrictive role in the activation of immunity against RBCs in dendritic cells, CD47 does not seem to have a significant effect on the antigen-presenting function of neutrophils in this context. Overall, these findings reveal that besides their classical antimicrobial role, neutrophils show plasticity in their phenotype.

Extensive Ethnic Variation and Linkage Disequilibrium at the FCGR2/3 Locus: Different Genetic Associations Revealed in Kawasaki Disease.

The human Fc-gamma receptors (FcγRs) link adaptive and innate immunity by binding immunoglobulin G (IgG). All human low-affinity FcγRs are encoded by the FCGR2/3 locus containing functional single nucleotide polymorphisms (SNPs) and gene copy number variants. This locus is notoriously difficult to genotype and high-throughput methods commonly used focus on only a few SNPs. We performed multiplex ligation-dependent probe amplification for all relevant genetic variations at the FCGR2/3 locus in >4,000 individuals to define linkage disequilibrium (LD) and allele frequencies in different populations. Strong LD and extensive ethnic variation in allele frequencies was found across the locus. LD was strongest for the FCGR2C-ORF haplotype (rs759550223+rs76277413), which leads to expression of FcγRIIc. In Europeans, the FCGR2C-ORF haplotype showed strong LD with, among others, rs201218628 (FCGR2A-Q27W, r2 = 0.63). LD between these two variants was weaker (r2 = 0.17) in Africans, whereas the FCGR2C-ORF haplotype was nearly absent in Asians (minor allele frequency <0.005%). The FCGR2C-ORF haplotype and rs1801274 (FCGR2A-H131R) were in weak LD (r2 = 0.08) in Europeans. We evaluated the importance of ethnic variation and LD in Kawasaki Disease (KD), an acute vasculitis in children with increased incidence in Asians. An association of rs1801274 with KD was previously shown in ethnically diverse genome-wide association studies. Now, we show in 1,028 European KD patients that the FCGR2C-ORF haplotype, although nearly absent in Asians, was more strongly associated with susceptibility to KD than rs1801274 in Europeans. Our data illustrate the importance of interpreting findings of association studies concerning the FCGR2/3 locus with knowledge of LD and ethnic variation.

FcγRIIIb Restricts Antibody-Dependent Destruction of Cancer Cells by Human Neutrophils.

The function of the low-affinity IgG-receptor FcγRIIIb (CD16b), which is uniquely and abundantly expressed on human granulocytes, is not clear. Unlike the other Fcγ receptors (FcγR), it is a glycophosphatidyl inositol (GPI) -anchored molecule and does not have intracellular signaling motifs. Nevertheless, FcγRIIIb can cooperate with other FcγR to promote phagocytosis of antibody-opsonized microbes by human neutrophils. Here we have investigated the role of FcγRIIIb during antibody-dependent cellular cytotoxicity (ADCC) by neutrophils toward solid cancer cells coated with either trastuzumab (anti-HER2) or cetuximab (anti-EGFR). Inhibiting FcγRIIIb using CD16-F(ab')2 blocking antibodies resulted in substantially enhanced ADCC. ADCC was completely dependent on FcγRIIa (CD32a) and the enhanced ADCC seen after FcγRIIIb blockade therefore suggested that FcγRIIIb was competing with FcγRIIa for IgG on the opsonized target cells. Interestingly, the function of neutrophil FcγRIIIb as a decoy receptor was further supported by using neutrophils from individuals with different gene copy numbers of FCGR3B causing different levels of surface FcγRIIIb expression. Individuals with one copy of FCGR3B showed higher levels of ADCC compared to those with two or more copies. Finally, we show that therapeutic antibodies intended to improve FcγRIIIa (CD16a)-dependent natural killer (NK) cell ADCC due to the lack of fucosylation on the N-linked glycan at position N297 of the IgG1 heavy chain Fc-region, show decreased ADCC as compared to regularly fucosylated antibodies. Together, these data confirm FcγRIIIb as a negative regulator of neutrophil ADCC toward tumor cells and a potential target for enhancing tumor cell destruction by neutrophils.

Genetic variation of human neutrophil Fcγ receptors and SIRPα in antibody-dependent cellular cytotoxicity towards cancer cells.

The efficacy of cancer therapeutic antibodies varies considerably among patients. Anti-cancer antibodies act through different mechanisms, including antibody-dependent cellular cytotoxicity (ADCC) triggered via Fcγ receptors (FcγR). This phagocyte ADCC can be promoted by interference with CD47-SIRPα interactions, but the magnitude of this enhancement also varies among individuals. Both FcγR and SIRPα display considerable genetic variation, and we investigated whether this explains some of the variability in ADCC. Because of linkage disequilibrium between FcγR variants the interpretation of previous reports suggesting a potential link between FcγR polymorphisms and ADCC has been troublesome. We performed an integrated genetic analysis that enables stratification. ADCC by activated human neutrophils towards Trastuzumab-coated breast cancer cells was predominantly dependent on FcγRIIa. Neutrophils from individuals with the FcγRIIa-131H polymorphic variant displayed significantly higher killing capacity relative to those with FcγRIIa-131R. Furthermore, ADCC was consistently enhanced by targeting CD47-SIRPα interactions, and there were no significant functional differences between the two most prevalent SIRPα polymorphic variants. Thus, neutrophil ADCC capacity is directly related to the FcγRIIa polymorphism, and targeting CD47-SIRPα interactions enhances ADCC independently of FcγR and SIRPα genotype, thereby further suggesting that CD47-SIRPα interference might be a generic strategy for potentiating the efficacy of antibody therapy in cancer.

Factor H-Related (FHR)-1 and FHR-2 Form Homo- and Heterodimers, while FHR-5 Circulates Only As Homodimer in Human Plasma.

The complement factor H-related (FHR) proteins are hypothesized to fine-tune the regulatory role of complement factor H (FH) in the alternative pathway of the complement system. Moreover, FHR-1, FHR-2, and FHR-5 have been proposed to be dimers, which further complicates accurate analysis. As FHRs are highly similar among themselves and toward FH, obtaining specific reagents for quantification of serum levels and functional analysis is challenging. In this study, we generated antibodies and developed ELISAs to measure FHR-1, FHR-2, and FHR-5 in serum. We used both recombinant and serum-derived proteins to show that four dimers occur in human circulation: homodimers of FHR-1, FHR-2, and FHR-5, as well as FHR-1/FHR-2 heterodimers. Heterodimers containing FHR-5 were not found. In individuals with homozygous CFHR1 deletions or compound heterozygous CFHR2 missense/nonsense mutations identified in this study, the respective FHR-1 and FHR-2 homo- and heterodimers were absent. Using FRET, we found that recombinant FHR dimers exchange monomers rapidly. This was confirmed ex vivo, using FHR-1- and FHR-2-deficient sera. Of all FHR dimers, FHR-5/5 homodimers demonstrated strong binding affinity toward heparin. Specific ELISAs demonstrated that serum levels of FHR-1/1, FHR-1/2, FHR-2/2, and FHR-5/5 dimers were low compared to FH, which circulates at a 10- to 200-fold molar excess. In summary, FHR-1, FHR-2, and FHR-5 homodimerize, with FHR-1 and FHR-2 forming heterodimers as well, and equilibrate quickly in plasma.

Mutation in an exonic splicing enhancer site causing chronic granulomatous disease.

In a male patient suffering from X-linked chronic granulomatous disease (CGD) we found a c.389G>T mutation in exon 5 of the CYBB gene. We have analyzed why 95% of the transcripts of this gene lacked exon 5, leading to a frameshift and premature termination codon. The mutation was located in a region comprising three putative exonic splicing enhancer binding sites, for SRSF1, SRFS2 and SRFS6, according to the ESEfinder Tool (http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi). With the Analyser Splice Tool we calculated the probability of skipping of exon 5 in CYBB mRNA, and by means of Sroogle the number of putative binding motifs for splicing enhancer and splicing silencer proteins (http://astlab.tau.ac.il/index.php). These analyses clarify why this exon was skipped in the majority of the mRNA. The normally spliced transcript contains an amino acid change p.Arg130Leu. This poorly expressed transcript gives rise to a protein with low expression but presumably normal activity, leading to a respiratory burst activity in the patient's neutrophils of about 15% of normal.

Nonclassical FCGR2C haplotype is associated with protection from red blood cell alloimmunization in sickle cell disease.

Red blood cell (RBC) transfusions are of vital importance in patients with sickle cell disease (SCD). However, a major complication of transfusion therapy is alloimmunization. The low-affinity Fcγ receptors, expressed on immune cells, are important regulators of antibody responses. Genetic variation in FCGR genes has been associated with various auto- and alloimmune diseases. The aim of this study was to evaluate the association between genetic variation of FCGR and RBC alloimmunization in SCD. In this case-control study, DNA samples from 2 cohorts of transfused SCD patients were combined (France and The Netherlands). Cases had a positive history of alloimmunization, having received ≥1 RBC unit. Controls had a negative history of alloimmunization, having received ≥20 RBC units. Single nucleotide polymorphisms and copy number variation of the FCGR2/3 gene cluster were studied in a FCGR-specific multiplex ligation-dependent probe amplification assay. Frequencies were compared using logistic regression. Two hundred seventy-two patients were included (130 controls, 142 cases). The nonclassical open reading frame in the FCGR2C gene (FCGR2C.nc-ORF) was strongly associated with a decreased alloimmunization risk (odds ratio [OR] 0.26, 95% confidence [CI] 0.11-0.64). This association persisted when only including controls with exposure to ≥100 units (OR 0.30, CI 0.11-0.85) and appeared even stronger when excluding cases with Rh or K antibodies only (OR 0.19, CI 0.06-0.59). In conclusion, SCD patients with the FCGR2Cnc-ORF polymorphism have over a 3-fold lower risk for RBC alloimmunization in comparison with patients without this mutation. This protective effect was strongest for exposure to antigens other than the immunogenic Rh or K antigens.

Hermansky-Pudlak syndrome type 2: Aberrant pre-mRNA splicing and mislocalization of granule proteins in neutrophils.

Hermansky-Pudlak syndrome type 2 (HPS2) is a syndrome caused by mutations in the beta-3A subunit of the adaptor protein (AP)-3 complex (AP3B1 gene). We describe five unreported cases with four novel mutations, one of which caused aberrant pre-mRNA splicing. A point mutation c.2702C>G in exon 23 of the AP3B1 gene caused deletion of 112 bp in the mRNA in two siblings. This mutation activates a cryptic donor splice site that overrules the wild-type donor splice site of this exon. Three other novel mutations in AP3B1 were identified, that is, a nonsense mutation c.716G>A (p.Trp239Ter), a 1-bp and a 4-bp deletion c.177delA and c.1839_1842delTAGA, respectively, both causing frameshift and premature termination of translation. Mass spectrometry in four of these HPS2 patients demonstrated the (near) absence of all AP-3 complex subunits. Immunoelectron microscopy on the neutrophils of two of these patients showed abnormal granule formation. We found clear mislocalization of myeloperoxidase in the neutrophils even though the content of this protein but not the activity seemed to be present at normal levels. In sum, HPS2 is the result of the absence of the entire AP-3 complex, which results in severe neutropenia with a defect in granule formation as the major hematological finding.