Diagnostics of Primary Immunodeficiencies through Next-Generation Sequencing.

BACKGROUND: Recently, a growing number of novel genetic defects underlying primary immunodeficiencies (PIDs) have been identified, increasing the number of PID up to more than 250 well-defined forms. Next-generation sequencing (NGS) technologies and proper filtering strategies greatly contributed to this rapid evolution, providing the possibility to rapidly and simultaneously analyze large numbers of genes or the whole exome. OBJECTIVE: To evaluate the role of targeted NGS and whole exome sequencing (WES) in the diagnosis of a case series, characterized by complex or atypical clinical features suggesting a PID, difficult to diagnose using the current diagnostic procedures. METHODS: We retrospectively analyzed genetic variants identified through targeted NGS or WES in 45 patients with complex PID of unknown etiology. RESULTS: Forty-seven variants were identified using targeted NGS, while 5 were identified using WES. Newly identified genetic variants were classified into four groups: (I) variations associated with a well-defined PID, (II) variations associated with atypical features of a well-defined PID, (III) functionally relevant variations potentially involved in the immunological features, and (IV) non-diagnostic genotype, in whom the link with phenotype is missing. We reached a conclusive genetic diagnosis in 7/45 patients (~16%). Among them, four patients presented with a typical well-defined PID. In the remaining three cases, mutations were associated with unexpected clinical features, expanding the phenotypic spectrum of typical PIDs. In addition, we identified 31 variants in 10 patients with complex phenotype, individually not causative per se of the disorder. CONCLUSION: NGS technologies represent a cost-effective and rapid first-line genetic approach for the evaluation of complex PIDs. WES, despite a moderate higher cost compared to targeted, is emerging as a valuable tool to reach in a timely manner, a PID diagnosis with a considerable potential to draw genotype-phenotype correlation. Nevertheless, a large fraction of patients still remains without a conclusive diagnosis. In these patients, the sum of non-diagnostic variants might be proven informative in future studies with larger cohorts of patients.

Impairment of dendritic cell functions in patients with adaptor protein-3 complex deficiency.

Hermansky-Pudlak syndrome type 2 (HPS2) is a primary immunodeficiency due to adaptor protein-3 (AP-3) complex deficiency. HPS2 patients present neutropenia, partial albinism, and impaired lysosomal vesicles formation in hematopoietic cells. Given the role of dendritic cells (DCs) in the immune response, we studied monocyte-derived DCs (moDCs) and plasmacytoid DCs (pDCs) in two HPS2 siblings. Mature HPS2 moDCs showed impaired expression of CD83 and DC-lysosome-associated membrane protein (LAMP), low levels of MIP1-ß/CCL4, MIG/CXCL9, and severe defect of interleukin-12 (IL-12) secretion. DCs in lymph-node biopsies from the same patients showed a diffuse cytoplasm reactivity in a large fraction of DC-LAMP(+) cells, instead of the classical dot-like stain. In addition, analysis of pDC-related functions of blood-circulating mononuclear cells revealed reduced interferon-α secretion in response to herpes simplex virus-1 (HSV-1), whereas granzyme-B induction upon IL-3/IL-10 stimulation was normal. Finally, T-cell costimulatory activity, as measured by mixed lymphocyte reaction assay, was lower in patients, suggesting that function and maturation of DCs is abnormal in patients with HPS2.

Failure of interferon-γ pre-treated mesenchymal stem cell treatment in a patient with Crohn's disease.

Mesenchymal stem cells (MSC) are cells of stromal origin which exhibit unlimited self-renewal capacity and pluripotency in vitro. It has recently been observed that MSC may also exert a profound immunosuppressive and anti-inflammatory effect both in vitro and in vivo with consequent potential use in autoimmune disorders. We present the case of a patient suffering from childhood-onset, multidrug resistant and steroid-dependent Crohn's disease who underwent systemic infusions of MSC, which led to a temporary reduction in CCR4, CCR7 and CXCR4 expression by T-cells, and a temporary decrease in switched memory B-cells, In addition, following MSC infusion, lower doses of steroids were needed to inhibit proliferation of the patient's peripheral blood mononuclear cells. Despite these changes, no significant clinical benefit was observed, and the patient required rescue therapy with infliximab and subsequent autologous hematopoietic stem cell transplantation. The results of biological and in vitro observations after MSC use and the clinical effects of infusion are discussed, and a brief description is provided of previous data on MSC-based therapy in autoimmune disorders.

Clinical, laboratory and molecular signs of immunodeficiency in patients with partial oculo-cutaneous albinism.

Hypopigmentation disorders that are associated with immunodeficiency feature both partial albinism of hair, skin and eyes together with leukocyte defects. These disorders include Chediak Higashi (CHS), Griscelli (GS), Hermansky-Pudlak (HPS) and MAPBP-interacting protein deficiency syndromes. These are heterogeneous autosomal recessive conditions in which the causal genes encode proteins with specific roles in the biogenesis, function and trafficking of secretory lysosomes. In certain specialized cells, these organelles serve as a storage compartment. Impaired secretion of specific effector proteins from that intracellular compartment affects biological activities. In particular, these intracellular granules are essential constituents of melanocytes, platelets, granulocytes, cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Thus, abnormalities affect pigmentation, primary hemostasis, blood cell counts and lymphocyte cytotoxic activity against microbial pathogens. Among eight genetically distinct types of HPS, only type 2 is characterized by immunodeficiency. Recently, a new subtype, HPS9, was defined in patients presenting with immunodeficiency and oculocutaneous albinism, associated with mutations in the pallidin-encoding gene, PLDN.Hypopigmentation together with recurrent childhood bacterial or viral infections suggests syndromic albinism. T and NK cell cytotoxicity are generally impaired in patients with these disorders. Specific clinical and biochemical phenotypes can allow differential diagnoses among these disorders before molecular testing. Ocular symptoms, including nystagmus, that are usually evident at birth, are common in patients with HPS2 or CHS. Albinism with short stature is unique to MAPBP-interacting protein (MAPBPIP) deficiency, while hemophagocytic lymphohistiocytosis (HLH) mainly suggests a diagnosis of CHS or GS type 2 (GS2). Neurological disease is a long-term complication of CHS, but is uncommon in other syndromic albinism. Chronic neutropenia is a feature of HPS2 and MAPBPIP-deficiency syndrome, whereas it is usually transient in CHS and GS2. In every patient, an accurate diagnosis is required for prompt and appropriate treatment, particularly in patients who develop HLH or in whom bone marrow transplant is required. This review describes the molecular and pathogenetic mechanisms of these diseases, focusing on clinical and biochemical aspects that allow early differential diagnosis.

Heterogeneous expression of toll-like receptors in lymphatic endothelial cells derived from different tissues.

As lymphatic endothelial cells (LECs) express different lymphatic and vascular markers depending on the organ they are derived from, we analysed whether they also show a heterogeneity of response against pathogens. To this end we analysed, for the presence of mRNA encoding for all human toll-like receptor (TLR), LECs isolated from lymph nodes and thymuses. RNA for TLR1-6 and 9 was identified in thymus-derived cells, whereas cells derived from lymph nodes contained mRNA for TLR1-4, 6 and 9, but failed to express mRNA specific for TLR5. The differential expression of TLRs was confirmed by the phosphorylation of nuclear factor-κB p65 only when the two types of LECs were incubated with the appropriate TLR agonists. The stimulation with specific agonists gives rise to a heterogeneous pattern of cytokine and chemokine secretion: thymus-derived LECs produced preferentially interleukin-6, interferon-inducible protein (IP)-10 and tumour necrosis factor-α, whereas cells prepared from lymph nodes mainly released interleukin-8, monocyte chemotactic protein-1, RANTES and (IP)-10. Finally, cells purified from lymph nodes expressed a higher level of intercellular adhesion molecule-1 than did cells prepared from the thymus when stimulated with several TLR agonists. The expression of a large set of TLRs and the responsiveness to specific agonists suggest that LECs are able to respond to pathogens, and the observed differences reflect specialized functions, redundancy and/or roles of LECs of different origin.

U94 of human herpesvirus 6 inhibits in vitro angiogenesis and lymphangiogenesis.

Human herpesvirus 6 (HHV-6) is a lymphotropic virus, but recent observations showed that also vascular endothelial cells (ECs) are susceptible to infection, both in vivo and in vitro. The observation that lymph nodes are a site of viral persistence suggests that lymphatic ECs (LECs) might be even more relevant for HHV-6 biology than vascular ECs. Here, we provide evidence that HHV-6 can infect LECs in vitro and establish a latent infection. Thus HHV-6 infection induces the loss of angiogenic properties both in LECs and in vascular ECs, as shown by the inability to form capillary-like structures and to seal wound scratches. The antiangiogenic effects observed in infected cells are associated to the expression of HHV-6 U94/rep, a latency-associated gene. In fact, transfection of U94/rep or addition of recombinant U94/REP protein to ECs inhibits the formation of in vitro capillary-like structures, reduces migration of ECs, and blocks angiogenesis, rendering rat aortic rings insensitive to VEGF-induced vasculogenetic activity. The ability of U94/rep to block different angiogenetic steps may lead to approaches in the potential control of the proliferation of blood and lymphatic vessels.