Unravelling novel genetic and functional mechanisms of primary immunodeficiencies

RESUMO: As imunodeficiências primárias (IDP) constituem um grupo heterogéneo de doenças congénitas que podem afetar o desenvolvimento e/ou a função de qualquer componente do sistema imunitário. Isto pode conduzir à alteração de qualquer uma das funções do sistema imune e portanto os doentes com IDP podem apresentar infeções recorrentes ou susceptibilidade especifica a determinado micro-organismo , mas também podem apresentar auto-imunidade, linfoproliferação, híper-inflamação ou até neoplasias. A recente generalização da disponibilidade das técnicas de sequenciação de nova geração permitu não só a identificação de genes responsáveis por IDP previamente descritas, mas também a descoberta de novas IDP. É sabido que a grande maioria das IDP são defeitos monogénicos que podem apresentar expressão e penetrância incompletas. O reconhecimento que mutações no mesmo gene podem conduzir a diferentes fenótipos, mas também que mutações em múltiplos genes diferentes podem conduzir ao mesmo fenótipo, tornou mandatória a utilização de técnicas de sequenciação de nova geração para elucidar os quadros clínicos destes doentes ou grupos de doentes. Ao compreendermos o impacto destas experiências da Natureza no sistema imunitário, foi possível não só expandir o conhecimento, mas também mudar condutas terapêuticas e o aconselhamento das famílias. Esta mudança de paradigma conduziu a uma necessidade continua de redefinir os sinais de alarme para a suspeição de IDP. Além da clássica susceptibilidade infeciosa, as manifestações auto-imunes de início precoce, a poliautoimunidade, a hiper-inflamação ou a infalamação recorrente (sistémica ou específica de órgão), a alergia exuberante (especialmente associada a outras manifestações), e até algumas neoplasias (como a leucemia juvenil mieolomonocítica) podem ser manifestações reveladoras de IDP. Esta tese teve como objectivo revelar novos mecanismos genéticos e/ou funcionais de IDPs. Para isso foram investigados doentes seguidos na Unidade de Imunodeficiências Primárias do Hospital Dona Estefânia, Centro Hospitalar de Lisboa Central, que tivessem uma IDP com sintomas incomuns e fatais ou uma IDP com sintomas atípicos que não permitisse estabelecer um diagnóstico genético definitivo. A investigação destes doentes permitiu adicionar novos fenótipos a IDP previamente conhecidas: hiperplasia nodular regenerativa num doente com imunodeficiência combinada grave causada por uma nova mutação hipomórfica no gene IL2RG; síndrome hemofagocítico secundário a infeções por BCG e Citomegalovirus num doente com deficiência do receptor do IFNγ; doença inflamatória intestinal e um fenótipo assemelhando-se ao sindrome trico-enterohepático causado por uma mutação hipomórfica do gene TTC7A; cutis laxa num doente com síndrome de APLAID causado por uma nova mutação do gene PLCG2. A investigação de um conjunto de doentes com doença inflamatória intestinal de início infantil (IO-IBD) permitiu a identificação de uma causa monogénica para a doença em 54% dos doentes e em muitos desses casos, isto conduziu a alterações terapêuticas. Pudemos descrever uma nova causa de IO-IBD, imunodeficiência e surdez neurossensorial, causada por mutações em STXBP3, que não tinha sido previamente associada a doença humana. Adicionalmente, num doente com um fenótipo IPEX-like, identificamos uma mutação homozigótica do gene ZC3H12A, codificando uma proteína Regnase-1 disfuncional condicionando uma regulação pós-transcricional aberrante de IL-6. Finalmente, numa doente com fasceites graves recorrentes, identificamos uma variante de significado incerto no gene IRG1 e provamos que essa variante conduz a uma alteração do metabolismo de ácido itacónico nos macrófagos, causando incapacidade de detectar adequadamente a resolução da infeção e conduzindo a hiperinflamação.ABSTRACT: Primary immunodeficiencies (PIDs) constitute a heterogeneous group of congenital diseases that affect the development and/or the function of any component of the immune system. As any function of the immune system can be affected, patients with PIDs can present with recurrent infections or a higher specific susceptibility to certain microorganisms, but also with autoimmunity, lymphoproliferation, hyper-inflammation or even cancer. With the massive generalization of the next-generation sequencing (NGS) techniques, the discovery of new PIDs and novel genetic causes for previously known PIDs has been boosted. It is known that the vast majority of PIDs are monogenic, with variable expressivity and often with incomplete penetrance. The recognition that mutations in the same gene can present with several clinical phenotypes, but also that mutations in multiple different genes can manifest with the same phenotype, has made mandatory the use of NGS to elucidate the clinical picture of individual patients or patient groups. By understanding the impact of such nature's experiments on the immune system, it has been possible to expand knowledge, change therapeutic strategies and family counselling. This has led to a continuous change of the warning signs that can reveal PIDs. Adding to the infectious susceptibility, early onset autoimmune manifestations, polyautoimmunity, hyper-inflammation, or recurrent inflammation (systemic or organspecific), exuberant allergy (especially if associated with other manifestations), and even some neoplasms (such as juvenile myelomonocyte leukaemia), could be indications to investigate the presence of a PID. This thesis aimed to reveal new genetic and/or functional mechanisms of PIDs. Patients enrolled in this thesis were followed at the Primary Immunodeficiencies Unit of Hospital Dona Estefânia, Centro Hospitalar de Lisboa Central, having either a known PID that presented fatal and uncommon symptoms or an undiagnosed PID presenting atypical clinical symptoms. The investigation of these patients added new phenotypes to previously described PIDs: nodular regenerative hyperplasia in a patient with leaky X-linked severe combined immunodeficiency caused by a novel hypomorphic mutation in the IL2RG gene; hemophagocytic lymphohistiocytosis secondary to BCG and Cytomegalovirus infections in a patient with IFNγ receptor deficiency; inflammatory bowel disease (IBD) and a phenotype resembling tricho-entero-hepatic syndrome caused by a hypomorphic TTC7A mutation; cutis laxa in an APLAID patient caused by a novel mutation in the PLCG2 gene. The investigation of an Infantile-onset IBD cohort of patients allowed the identification of a monogenic cause for the disease in 54% of the patients and in many cases, this led to changes in the therapeutic approach. We were able to identify a novel cause of IO-IBD, immunodeficiency and sensoryneural immunodeficiency, caused by STXBP3 mutations, not previously associated with human disease. Moreover, in a patient presenting an IPEX-like phenotype we have identified a novel PID caused by homozygous mutations in the ZC3H12A gene, leading to a dysfunctional regnase-1 protein that caused aberrant post-transcriptional regulation of IL-6 production. Finally, in a patient with severe recurrent fasciitis we identified a variant of unknown significance in the IRG1 gene and aimed to prove that the IRG1 variant lead to altered itaconate metabolism in macrophages, thus resulting in inappropriate sensing of the resolution of the infection and hyperinflammation

ERITRODERMIA: PRIMEIRA MANIFESTAÇÃO DE DÉFICE IMUNITÁRIO CONGÉNITO

Transfusion-associated graft-versus-host disease (TA-GVHD) is a rare complication of transfusion of nonirradiated blood components. It usually affects children in high-risk groups, including those who have primary immunodeficiencies (PIDs). It usually presents with skin, hepatic, digestive, and hematologic involvement and is normally fatal. We report a case of a nonlethal, attenuated, TA-GVHD which was the clue to the diagnosis of combined immunodeficiency. The disease was marked by the presence of a severe rash but lacked all the other usual manifestations and wasn’t fatal due to the fact that this child was under high-dose corticotherapy. This led to the survival of this child and allowed the diagnosis of a combined immunodeficiency. The definitive diagnosis of GVHD can be problematic because the clinical and histological features can mimic other conditions such as drug eruptions, viral rash or eczema.A Doença do Enxerto Contra o Hospedeiro associada a transfusão (DEH-AT) é uma complicação rara da transfusão de hemoderivados não irradiados que afecta habitualmente indivíduos pertencentes a grupo de risco, nomeadamente crianças com imunodeficiência congénita. Caracteriza-se por um envolvimento cutâneo, gastrointestinal e hematológico, sendo habitualmente fatal. Descreve-se um caso DEH-AT atenuada que constituiu o indício para o diagnóstico de uma imunodeficiência combinada. A doença manifestou-se com rash característico mas não apresentou envolvimento digestivo ou hematológico, provavelmente pela corticoterapia concomitante. Tal facto permitiu a sobrevivência da criança e o diagnóstico de uma imunodeficência combinada. O diagnóstico definitivo de DEH-AT pode ser difícil uma vez que as manifestações clínicas e histológicas mimetizam, com frequência, outras situações, tais como toxidermia ou doença viral

H Factor Deficiency: A Case with an Atypical Presentation

We report a case of an 18-month-old boy with H factor deficiency with atypical presentation: recurrent acute otitis media and several maternal family members with autoimmune disorders (vitiligo, thyroiditis and immune trombocytopenia). Blood tests revealed low C3 and AH50, as well as low properdin and H factor. I factor was normal. CFH gene molecular test confirmed the H factor deficiency diagnosis. This child had none of the typical manifestations of this disorder, namely Neisseria meningitidis infection or renal disease (glomerulonephritis and atypical haemolytic uremic syndrome). Autoimmune family history and correct interpretation of blood tests’ results were crucial for this diagnosis

Recurrent pyogenic infections caused by a novel Gln1420* mutation in the C3 gene

Funding Information: The present publication was funded by Fundação Ciência e Tecnologia, IP national support through CHRC (UIDP/04923/2020).C3 is a crucial protein of the complement system. Congenital C3 deficiency is extremely rare and manifests through recurrent, severe infections and should always be considered as a differential diagnosis of recurrent pyogenic infections. We report a case of a patient with a novel C3 gene mutation, responsible for complete C3 deficiency with impaired complement system activation and recurrent infections.publishersversionpublishe

Primary Immunodeficiencies, Massive EBV+ T-Cell Lympoproliferation Leading to the Diagnosis of ICF2 Syndrome

Funding: The present publication was funded by Fundação Ciência e Tecnologia, IP national support through CHRC (UIDP/04923/2020)In immunocompromised patients, EBV may elicit B-cell transformation and proliferation. A 5-year-old microcephalic boy was admitted with fever and non-malignant polymorphic T-cell lymphoproliferative disease associated with EBV. A presumptive diagnosis of primary immunodeficiency with inability to control EBV was made and next-generation sequencing led to the identification of a novel ZBTB24 mutation (ICF2-syndrome). This case shows that susceptibility to EBV seems to be particular of ICF-2 as it has not been described in the other types of ICF. It is mandatory to raise the hypothesis of an underlying PID in case of severe EBV infection.publishersversionpublishe

Novel PLCG2 Mutation in a Patient With APLAID and Cutis Laxa.

Background: The auto-inflammation and phospholipase Cγ2 (PLCγ2)-associated antibody deficiency and immune dysregulation (APLAID) syndrome is a rare primary immunodeficiency caused by a gain-of-function mutation S707Y in the PLCG2 gene previously described in two patients from one family. The APLAID patients presented with early-onset blistering skin lesions, posterior uveitis, inflammatory bowel disease (IBD) and recurrent sinopulmonary infections caused by a humoral defect, but lacked circulating autoantibodies and had no cold-induced urticaria, contrary to the patients with the related PLAID syndrome. Case: We describe a new APLAID patient who presented with vesiculopustular rash in the 1st weeks of life, followed by IBD, posterior uveitis, recurrent chest infections, interstitial pneumonitis, and also had sensorineural deafness and cutis laxa. Her disease has been refractory to most treatments, including IL1 blockers and a trial with ruxolitinib has been attempted. Results: In this patient, we found a unique de novo heterozygous missense L848P mutation in the PLCG2 gene, predicted to affect the PLCγ2 structure. Similarly to S707Y, the L848P mutation led to the increased basal and EGF-stimulated PLCγ2 activity in vitro. Whole blood assays showed reduced production of IFN-γ and IL-17 in response to polyclonal T-cell stimulation and reduced production of IL-10 and IL-1β after LPS stimulation. Reduced IL-1β levels and the lack of clinical response to treatment with IL-1 blockers argue against NLRP3 inflammasome hyperactivation being the main mechanism mediating the APLAID pathogenesis. Conclusion: Our findings indicate that L848P is novel a gain-of-function mutation that leads to PLCγ2 activation and suggest cutis laxa as a possible clinical manifestations of the APLAID syndrome

Hemophagocytic lymphohistiocytosis in an adolescent with NLRP12-related autoinflammatory disorder—A case report

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Characterization of the clinical and immunologic phenotype and management of 157 individuals with 56 distinct heterozygous NFKB1 mutations

Background: An increasing number of NFKB1 variants are being identified in patients with heterogeneous immunologic phenotypes. Objective: To characterize the clinical and cellular phenotype as well as the management of patients with heterozygous NFKB1 mutations. Methods: In a worldwide collaborative effort, we evaluated 231 individuals harboring 105 distinct heterozygous NFKB1 variants. To provide evidence for pathogenicity, each variant was assessed in silico; in addition, 32 variants were assessed by functional in vitro testing of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-kappa B) signaling. Results: We classified 56 of the 105 distinct NFKB1 variants in 157 individuals from 68 unrelated families as pathogenic. Incomplete clinical penetrance (70%) and age-dependent severity of NFKB1-related phenotypes were observed. The phenotype included hypogammaglobulinemia (88.9%), reduced switched memory B cells (60.3%), and respiratory (83%) and gastrointestinal (28.6%) infections, thus characterizing the disorder as primary immunodeficiency. However, the high frequency of autoimmunity (57.4%), lymphoproliferation (52.4%), noninfectious enteropathy (23.1%), opportunistic infections (15.7%), autoinflammation (29.6%), and malignancy (16.8%) identified NF-kappa B1-related disease as an inborn error of immunity with immune dysregulation, rather than a mere primary immunodeficiency. Current treatment includes immunoglobulin replacement and immunosuppressive agents. Conclusions: We present a comprehensive clinical overview of the NF-kappa B1-related phenotype, which includes immunodeficiency, autoimmunity, autoinflammation, and cancer. Because of its multisystem involvement, clinicians from each and every medical discipline need to be made aware of this autosomal-dominant disease. Hematopoietic stem cell transplantation and NF-kappa B1 pathway-targeted therapeutic strategies should be considered in the future.Peer reviewe