UNIVERSITY OF NAPLES “FEDERICO II” SCHOOL OF MEDICINE AND SURGERY DEPARTMENT OF TRANSLATIONAL MEDICAL SCIENCES PhD Program “Human Reproduction, Development and Growth” Director Prof. Claudio Pignata PhD Thesis Novel strategies in the approach to primary immunodeficiencies to discover new pathogenic mechanisms and complex clinical phenotypes Student Tutor Dr. Giuliana Giardino Prof. Claudio Pignata Academic Year 2015-2016 1 BACKGROUND AND AIM .................................................................................................................... 4 CHAPTER I ............................................................................................................................................ 12 “Targeted Next Generation Sequencing (TNGS): a powerfull tool for a rapid diagnosis of PIDs” ....... 12 1.1 Targeted next generation sequencing revealed MYD88 deficiency in a child with chronic Yersiniosis and granulomatous .............................................................................................................. 13 1.2 Diagnostics of Primary Immunodeficiencies through targeted Next Generation Sequencing........ 25 CHAPTER 2 ........................................................................................................................................... 38 “STAT1 gain of function mutation in the pathogenesis of chronic mucocutaneous disease in the context of a complex multisystemic disorder” .................................................................................................... 38 2.1 Novel STAT1 gain of function mutation and suppurative infections................................................ 40 2.2 Heterozygous STAT1 gain-of-function mutations underlie a broad clinical phenotype: an international survey of 234 patients from 140 kindreds ...................................................................... 46 2.3 Clinical heterogeneity of dominant chronic mucocutaneous candidiasis disease: presenting as treatment-resistant candidiasis and chronic lung disease .................................................................... 63 CHAPTER 3 ........................................................................................................................................... 94 “Ectodermal disorders and PIDs” ........................................................................................................... 94 3.1 B cells from nuclear factor kB essential modulator deficient patients fail to differentiate to antibody secreting cells in response to TLR9 ligand. .......................................................................... 98 3.2 Insight into IKBKG/NEMO locus: report of new mutations and complex genomic rearrangements leading to incontinentia pigmenti disease. .............................................................. 105 3.3 Unraveling the Link Between Ectodermal Disorders and Primary Immunodeficiencies ............ 120 3.4 FOXN1 in organ development and human diseases. ......................................................................... 135 2 3.5 FOXN1: A Master Regulator Gene of Thymic Epithelial Development Program ........................ 150 CHAPTER 4 ......................................................................................................................................... 163 “Rare genetic syndrome involving immune system” ............................................................................ 163 4.1 Severe combined immunodeficiencies – an update .................................................................... 163 4.2 Phenotypic characterization and outcome of paediatric patients affected with haemophagocytic syndrome of unknown genetic cause ................................................................................................... 184 4.3 Intergenerational and intrafamilial phenotypic variability in 22q11.2 Deletion syndrome subjects 194 4.4 Gastrointestinal involvement in patients affected with 22q11.2 deletion syndrome. .................... 204 TECHNOLOGIES ................................................................................................................................ 211 REFERENCES...................................................................................................................................... 226 SUMMARY .......................................................................................................................................... 260 CURRICULUM VITAE ....................................................................................................................... 263 3 BACKGROUND AND AIM Primary immunodeficiency disorders (PIDs) are a group of inherited disorders of the immune system, resulting in an increased rate and severity of infection. Malignancy and immune dysregulation, the latter resulting in autoimmune disease and aberrant inflammatory responses may also feature these syndromes (1). PIDs differ from secondary immunodeficiencies occurring during certain viral infections, after immunosuppression, during treatment of systemic autoimmune disease, or in association with cancer chemotherapy (1). The overall prevalence of PIDs is about 1:2000 live births (2, 3) with an estimated prevalence of the severe forms of PIDs of 1:58000 live birth (4). The male/female ratio of PIDDs is approximately 5:1 in infants and children but approaches 1:1 in adults (2, 5). PIDs are classified according to mechanistic and clinical descriptive characteristics. In particular, according to the mechanistic characteristics, they may be divided in defects of innate immunity, disorders of immune dysregulation, autoinflammatory syndromes, phagocyte and complement system defects, or defect of adaptive immunity. The last group can be further subdivided into humoral deficiencies, and combined deficiencies affecting both humoral and cellular mechanisms. Recently, anticytokine autoantibodies have been identified in the pathophysiology of some Mendelian PIDs. PIDs characterized by antibody deficiency are the most frequent group, accounting for approximately half of all patients with PIDs (2, 6). Increased susceptibility to infections represent the main clinical feature of PIDs. The organ systems involved and the typical pathogens vary with the specific type of immune 4 defect (7, 8). Other features include autoimmune disease and malignancy, which may be often observed in a variety of immunodeficiencies (9, 10). Hypersensitivity to environmental and/or food allergens, may also feature a variety of PIDs (11). A careful identification of the foci of infections, of the organisms, and of the response to treatment are necessary to point the diagnosis through a specific PID. Any other conditions that might predispose to infection, including anatomic defects, allergy, and metabolic disorders, should be also ruled out (12). The clinical presentation usually guide the initial evaluation with a stepwise approach (screening tests followed by advanced tests) that ensures an efficient evaluation of mechanisms of immune dysfunction underlying the clinical presentation (2, 13). In some case, diagnosis may be difficult and may require costly sophisticated tests. First line laboratory assessment includes evaluation of serum immunoglobulin levels and leukocyte and lymphocyte subpopulations, and evaluation of the specific immune response including responses against vaccine antigens, or responses to natural exposure or infections. According to the severity of clinical manifestations combined immunodeficiencies are classified in severe combined immunodeficiency (SCID) or combined ID with a ‘‘less severe’’ clinical presentation (14). Patients with SCID show a complete absence of specific immunity and are prone to develop infections caused by the entire range of possible pathogens, including opportunistic organisms (15-17). SCID is a life threatning condition and diagnosis is an urgent medical condition since if not treated these infants can succumb to severe infection at any time. Definitive hematopoietic stem cell therapy (HSCT) should be 5 performed as soon as possible (18-22). A variety of additional genetic defects leading to impairment of T- and B-cell function have been described, including hyper-IgM Wiskott- Aldrich, DiGeorge, ataxia-telangiectasia, hyper-IgE syndromes, and others. Many of these diseases are characterized by peculiar clinical features that might influence or guide the diagnostic approach. The principal clinical manifestations of humoral immunodeficiency are recurrent bacterial infections of the upper and lower respiratory tract (23, 24). The disorders of immune dysregulation include the hemophagocytic syndromes, syndromes with autoimmunity and hypersensitivity, and lymphoproliferation. The hemophagocytic syndromes are characterized by fulminant acute presentations triggered by viral infections (25, 26). These patients usually require aggressive chemotherapy followed by HSCT to prevent immediate fatality (25, 26). Phagocytic cell defects can present with severe pyogenic bacterial and fungal infections of the respiratory tract, skin, and viscera and gingivostomatitis. Laboratory evaluation might show neutropenia, normal neutrophil numbers, or marked neutrophilia (mainly in cellular adhesion defects) (27, 28). Disorders of innate immunity are rare and include defects of Toll-like receptor (TLRs) signaling, such as nuclear factor kB essential modulator (NEMO) syndrome, often exhibiting ectodermal dysplasia along with infection susceptibility with a narrow (eg, predominantly pyogenic