Gigliola Di Matteo

Hematologically important mutations: X-linked chronic granulomatous disease (third update)

Chronic granulomatous disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. The disease is caused by a lack of superoxide production by the leukocyte enzyme NADPH oxidase. Superoxide is used to kill phagocytosed micro-organisms in neutrophils, eosinophils, monocytes and macrophages. The leukocyte NADPH oxidase is composed of five subunits, of which the enzymatic component is gp91-phox, also called Nox2. This protein is encoded by the CYBB gene on the X chromosome. Mutations in this gene are found in about 70% of all CGD patients. This article lists all mutations identified in CYBB in the X-linked form of CGD. Moreover, apparently benign polymorphisms in CYBB are also given, which should facilitate the recognition of future disease-causing mutations.

CD4 and Major Histocompatibility Complex Class I Downregulation by the Human Immunodeficiency Virus Type 1 Nef Protein in Pediatric AIDS Progression

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) nef gene is a crucial determinant in AIDS disease progression. Although several in vitro activities have been attributed to the Nef protein, identifying the one critical for in vivo pathogenicity remains elusive. In this study, we examined a large number of nef alleles derived at various time points from 13 perinatally infected children showing different progression rates: six nonprogressors (NPs), three slow progressors (SPs), and four rapid progressors (RPs). The patient-derived nef alleles were analyzed for their steady-state expression of a Nef protein, for their relative ability to downregulate cell surface expression of CD4 and major histocompatibility complex class I (MHC-I) and for their capacity to bind the clathrin adaptor AP-1 complex. We found that NP-derived nef alleles, compared to nef alleles isolated from SPs and RPs, had reduced CD4 and MHC-I downregulation activities. In contrast, SP- and RP-derived nef alleles did not differ and efficiently downregulated both CD4 and MHC-I. AP-1 binding was a conserved function of primary nef alleles not correlated with clinical progression. Defective Nef proteins from NPs, rather than sharing common specific changes in their sequences, accumulated various amino acid substitutions, mainly located outside the conserved domains previously associated with Nef biological properties. Our data indicate that Nef-mediated downregulation of cell surface CD4 and MHC-I significantly contributes to the expression of the pathogenic potential of HIV-1.

Structural defects and variations in the HIV-1 nef gene from rapid, slow and non-progressor children.

Objectives: Evaluation of sequence evolution as well as structural defects and mutations of the human immunodeficiency virus‐type 1 (HIV‐1) nef gene in relation to disease progression in infected children. Design: We examined a large number of nef alleles sequentially derived from perinatally HIV‐1‐infected children with different rates of disease progression: six non‐progressors (NPs), four rapid progressors (RPs), and three slow progressors (SPs). Methods: Nef alleles (182 total) were isolated from patients’ peripheral blood mononuclear cells (PBMCs), sequenced and analysed for their evolutionary pattern, frequency of mutations and occurrence of amino acid variations associated with different stages of disease. Results: The evolution rate of the nef gene apparently correlated with CD4+ decline in all progression groups. Evidence for rapid viral turnover and positive selection for changes were found only in two SPs and two RPs respectively. In NPs, a higher proportion of disrupted sequences and mutations at various functional motifs were observed. Furthermore, NP‐derived Nef proteins were often changed at residues localized in the folded core domain at cytotoxic T lymphocytes (CTL) epitopes (E105, K106, E110, Y132, K164, and R200), while other residues outside the core domain are more often changed in RPs (A43) and SPs (N173 and Y214). Conclusions: Our results suggest a link between nef gene functions and the progression rate in HIV‐1‐infected children. Moreover, non‐progressor‐associated variations in the core domain of Nef, together with the genetic analysis, suggest that nef gene evolution is shaped by an effective immune system in these patients.

Dual-regulated Lentiviral Vector for Gene Therapy of X-linked Chronic Granulomatosis

Regulated transgene expression may improve the safety and efficacy of hematopoietic stem cell (HSC) gene therapy. Clinical trials for X-linked chronic granulomatous disease (X-CGD) employing gammaretroviral vectors were limited by insertional oncogenesis or lack of persistent engraftment. Our novel strategy, based on regulated lentiviral vectors (LV), targets gp91(phox) expression to the differentiated myeloid compartment while sparing HSC, to reduce the risk of genotoxicity and potential perturbation of reactive oxygen species levels. Targeting was obtained by a myeloid-specific promoter (MSP) and posttranscriptional, microRNA-mediated regulation. We optimized both components in human bone marrow (BM) HSC and their differentiated progeny in vitro and in a xenotransplantation model, and generated therapeutic gp91(phox) expressing LVs for CGD gene therapy. All vectors restored gp91(phox) expression and function in human X-CGD myeloid cell lines, primary monocytes, and differentiated myeloid cells. While unregulated LVs ectopically expressed gp91(phox) in CD34(+) cells, transcriptionally and posttranscriptionally regulated LVs substantially reduced this off-target expression. X-CGD mice transplanted with transduced HSC restored gp91(phox) expression, and MSP-driven vectors maintained regulation during BM development. Combining transcriptional (SP146.gp91-driven) and posttranscriptional (miR-126-restricted) targeting, we achieved high levels of myeloid-specific transgene expression, entirely sparing the CD34(+) HSC compartment. This dual-targeted LV construct represents a promising candidate for further clinical development.

Chronic granulomatous disease: Clinical, molecular, and therapeutic aspects

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency caused by defects in the genes encoding any of the NADPH oxidase components responsible for the respiratory burst of phagocytic leukocytes. CGD is a genetically heterogeneous disease with an X‐linked recessive (XR‐CGD) form caused by mutations in the CYBB gene encoding the gp91phox protein, and an autosomal recessive (AR‐CGD) form caused by mutations in the CYBA, NCF1, NCF2, or NCF4 genes encoding p22phox, p47phox, p67phox, and p40phox, respectively. Patients suffering from this disease are susceptible to severe life‐threatening bacterial and fungal infections and excessive inflammation characterized by granuloma formation in any organ, for instance, the gastrointestinal and genitourinary tract. An early diagnosis of and the prompt treatment for these conditions are crucial for an optimal outcome of affected patients. To prevent infections, CGD patients should receive lifelong antibiotics and antifungal prophylaxis. These two measures, as well as newer more effective antimicrobials, have significantly modified the natural history of CGD, resulting in a remarkable change in overall survival, which is now around 90%, reaching well into adulthood. At present, hematopoietic stem cell transplantation (HSCT) is the only definitive treatment that can cure CGD and reverse organ dysfunction. Timing, donor selection, and conditioning regimens remain the key points of this therapy. In recent years, gene therapy (GT) for XR‐CGD has been proposed as an alternative to HSCT for CGD patients without a matched donor. After the failure of the first trials performed with retroviral vectors, some groups have proposed the use of regulated SIN‐lentiviral vectors targeting gp91phox expression in myeloid cells to increase the safety and efficacy of the GT protocols.

The case of an APDS patient: Defects in maturation and function and decreased in vitro anti-mycobacterial activity in the myeloid compartment

Activated PI3-kinase delta syndrome (APDS) was recently reported as a novel primary immunodeficiency caused by heterozygous gain-of-function mutations in PIK3CD gene. Here we describe immunological studies in a 19year old APDS patient for whom genetic diagnosis was discovered by Whole Exome Sequencing (WES) analysis. In addition to the progressive lymphopenia and defective antibody production we showed that the ability of the patients B cells to differentiate in vitro is severely reduced. An in depth analysis of the myeloid compartment showed an increased expression of CD83 activation marker on monocytes and mono-derived DC cells. Moreover, monocytes-derived macrophages (MDMs) failed to solve the Mycobacterium bovis bacillus Calmette Guèrin (BCG) infection in vitro. Selective p110δ inhibitor IC87114 restored the MDM capacity to kill BCG in vitro. Our data show that the constitutive activation of Akt-mTOR pathway induces important alterations also in the myeloid compartment providing new insights in order to improve the therapeutic approach in these patients.

Identification of a Btk mutation in a dysgammaglobulinemic patient with reduced B cells: XLA diagnosis or not?

The identification of a Btk mutation in a male patient with <2% CD19(+) B cells warrants making the diagnosis of X-linked Agammaglobulinemia (XLA). Herein we report the case of a 31 year-old male with a gradual decline of peripheral B lymphocytes and low IgA and IgM but normal IgG levels. His clinical history revealed recurrent respiratory and skin infections, sclerosing cholangitis and chronic obstructive pancreatitis. Molecular studies revealed a novel aminoacidic substitution in Btk protein (T316A). His mother, maternal aunts and a maternal female cousin were heterozygotes for the same Btk mutation and were variably affected with pulmonary emphysema. This is a puzzling case where the patients clinical history and laboratory findings divorce molecular genetics. Either this case confirms the variable expressivity of XLA disease or the T316A change in Btk SH2 domain is a novel non-pathogenic mutation and another unknown gene alteration is responsible for the disease.

Large Deletion of MAGT1 Gene in a Patient with Classic Kaposi Sarcoma, CD4 Lymphopenia, and EBV Infection

Abbreviations EBNA Epstein–Barr nuclear antigen EBV Epstein–Barr virus HE Hematoxylin eosin HHV-8 Human herpes virus 8 HIV Human immunodeficiency virus IVIG Intravenous immunoglobulin KS Kaposi Sarcoma MFI Mean fluorescence intensity PBMC Peripheral blood mononuclear cells PID Primary immunodeficiency PLCγ1 Phospholipase C gamma 1 TCR T cell receptor WES Whole exome sequencing WGS Whole genome sequencing XMEN X-linked immunodeficiency with magnesium defect, Epstein–Barr virus infection, and neoplasia

Natural killer cells from patients with recombinase-activating gene and non-homologous end joining gene defects comprise a higher frequency of CD56bright NKG2A+++ cells, and yet display increased degranulation and higher perforin content

Mutations of the recombinase-activating genes 1 and 2 (RAG1 and RAG2) in humans are associated with a broad range of phenotypes. For patients with severe clinical presentation, hematopoietic stem cell transplantation (HSCT) represents the only curative treatment; however, high rates of graft failure and incomplete immune reconstitution have been observed, especially after unconditioned haploidentical transplantation. Studies in mice have shown that Rag−/− natural killer (NK) cells have a mature phenotype, reduced fitness, and increased cytotoxicity. We aimed to analyze NK cell phenotype and function in patients with mutations in RAG and in non-homologous end joining (NHEJ) genes. Here, we provide evidence that NK cells from these patients have an immature phenotype, with significant expansion of CD56bright CD16−/int CD57− cells, yet increased degranulation and high perforin content. Correlation was observed between in vitro recombinase activity of the mutant proteins, NK cell abnormalities, and in vivo clinical phenotype. Addition of serotherapy in the conditioning regimen, with the aim of depleting the autologous NK cell compartment, may be important to facilitate engraftment and immune reconstitution in patients with RAG and NHEJ defects treated by HSCT.

Serratia marcescens osteomyelitis in a newborn with chronic granulomatous disease.

The Pediatric Infectious Disease Journal • Volume 32, Number 8, August 2013 unrelated bone marrow transplantation from a suitably matched donor. Osteomyelitis occurs in 13% of CGD patients; in childhood, most occur in the long bones, with only 1–4% in the hands or feet. Osteomyelitis in infancy particularly when caused by S. marcenscens should raise the suspicion of the presence of CGD as an underlying condition. We believe that this is the first case of osteomyelitis in a CGD patient reported in the neonatal period.