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1.
Artigo em Inglês | MEDLINE | ID: mdl-31775018

RESUMO

We describe the first case of MAN2B2 deficiency in a patient with immune dysregulation, developmental delay, and stroke. Altered mannosylation profile was restored in patient cells upon transduction of wild-type MAN2B2.

3.
Artigo em Inglês | MEDLINE | ID: mdl-31319217

RESUMO

BACKGROUND: Mastocytosis is a clonal mast cell disorder associated with elevated mast cell mediators, which themselves have been reported to affect lymphocyte function. However, the impact of an expanded mast cell compartment on lymphocyte subpopulations, and their correlation with clinical phenotypes in patients with indolent systemic mastocytosis (ISM), has not been explored. OBJECTIVE: To examine the immunophenotype of circulating lymphocytes in patients with ISM compared with healthy adult controls and examine relationships with aspects of clinical disease. METHODS: We examined lymphocyte subsets in 20 adult patients with ISM and 40 healthy adult volunteers by multiparameter flow cytometry. Results were correlated with clinical characteristics. RESULTS: Patients with ISM exhibited a significantly lower median frequency and absolute cell count of both circulating CD8+ T cells and natural killer cells accompanying a significantly increased ratio of CD4+/CD8+ T cells when compared with healthy volunteers. Stratification of our ISM patient cohort according to clinical manifestations revealed that CD19+CD21lowCD38low B cells were significantly higher in patients with a history of autoimmune disease and counts of terminally differentiated CD4+ T cells were significantly higher in patients with osteoporosis or osteopenia. CONCLUSIONS: Several circulating lymphocyte subpopulations in patients with ISM were significantly different when compared with healthy controls; in specific lymphocyte subsets, this lymphocyte skewing correlated with clinical observations including osteoporosis and autoimmune disease. These data suggest the need for further studies on abnormalities in lymphocyte subsets and the attendant clinical consequences in both mast cell proliferative and activation disorders.

4.
Front Immunol ; 10: 1193, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31191551

RESUMO

Autoimmune lymphoproliferative syndrome (ALPS) is caused by germline or somatic loss of function FAS mutations resulting in impaired apoptosis and consequent expansion of T-lymphocytes causing organomegaly and autoimmune anemia, neutropenia and thrombocytopenia. Herein, we report on a case of disseminated varicella zoster infection after post-partum vaccination in a patient found to have CD4 lymphopenia and eventually diagnosed with ALPS caused by a novel germline missense mutation in FAS death-domain. A subsequent retrospective analysis of 169 patients of the NIH ALPS-FAS cohort, revealed that CD4-T-cells lymphopenia (< 300 cells/µl) may occur in 5% of ALPS-FAS patients irrespectively of the underlying genetic defect, organomegaly or immunosuppressive treatment. Although immunophenotyping did not show depletion of specific CD4-T-cells subpopulations, CD4-lymphopenic ALPS-FAS subjects had an expansion of a subset of circulating T-follicular-helper (cTfh) cells, associated with autoantibody production (CCR7lowPD-1high). Furthermore, autoantibodies binding on CD4-T-cells were detected in 50% of the CD4-lymphopenic ALPS-FAS patients and caused cytotoxicity in a natural killer (NK)-mediated antibody-dependent-cellular cytotoxicity assay. Such autoantibodies can therefore be associated with CD4-T-cell death, impaired activation induced proliferation or impaired trafficking. The expansion of autoreactive T-cells in ALPS-FAS is known to be associated with autoimmune clinical manifestations, however our study reveals that ALPS-FAS can also be associated with a paradoxical depletion of CD4-T-cells due to the presence of autoantibodies on the surface of CD4-T-cells which can in turn result in increased susceptibility to opportunistic infections. These novel findings have implications for the diagnosis, clinical monitoring, and management of patients with ALPS-FAS.

6.
Front Immunol ; 10: 479, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30936877

RESUMO

Background: HOIP is the catalytic subunit of the linear ubiquitination chain assembly complex (LUBAC) that is essential for NF-κB signaling and thus proper innate and adaptive immunity. To date only one patient with HOIP deficiency has been reported with clinical characteristics that include autoinflammation, immunodeficiency, amylopectinosis, and systemic lymphangiectasia. Case: We sought to identify a genetic cause of a disease for an 8 year-old girl who presented with early-onset immune deficiency and autoinflammation. Methods: Targeted next generation sequencing of 352 immune-related genes was performed. Functional studies included transcriptome analysis, cytokine profiling, and protein analysis in patients' primary cells. Results: We identified biallelic variants in close proximity to splice sites (c.1197G>C and c.1737+3A>G) in the RNF31 gene. RNA extracted from patient cells showed alternatively spliced transcripts not present in control cells. Protein expression of HOIP and LUBAC was reduced in primary cells as shown by western blotting. Patient-derived fibroblasts demonstrated attenuated IL-6 production, while PBMCs showed higher TNF production after stimulation with proinflammatory cytokines. RNA sequencing of whole blood RNA and PBMCs demonstrated a marked transcriptome wide change including differential expression of type I interferon regulated genes. Conclusion: We report the second case of HOIP deficiency with novel compound heterozygous mutations in RNF31 and distinct clinical and molecular features. Our results expand on the clinical spectrum of HOIP deficiency and molecular signatures associated with LUBAC deficiency.

7.
Front Immunol ; 10: 77, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30891027

RESUMO

Phosphoinositide 3-kinase (PI3K) plays an integral role in lymphocyte function. Mutations in PIK3CD and PIK3R1, encoding the PI3K p110δ and p85α subunits, respectively, cause increased PI3K activity and result in immunodeficiency with immune dysregulation. We describe here the first cases of disseminated and congenital toxoplasmosis in a mother and child who share a pathogenic mutation in PIK3R1 and review the mechanisms underlying susceptibility to severe Toxoplasma gondii infection in activated PI3Kδ syndrome (APDS) and in other forms of primary immunodeficiency.

8.
Front Immunol ; 10: 23, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30778343

RESUMO

Severe combined immunodeficiency (SCID) represents one of the most severe forms of primary immunodeficiency (PID) disorders characterized by impaired cellular and humoral immune responses. Here, we report the clinical, immunological, and molecular findings in 57 patients diagnosed with SCID from India. Majority of our patients (89%) presented within 6 months of age. The most common clinical manifestations observed were recurrent pneumonia (66%), failure to thrive (60%), chronic diarrhea (35%), gastrointestinal infection (21%), and oral candidiasis (21%). Hematopoietic Stem Cell Transplantation (HSCT) is the only curative therapy available for treating these patients. Four patients underwent HSCT in our cohort but had a poor survival outcome. Lymphopenia (absolute lymphocyte counts/µL <2,500) was noted in 63% of the patients. Based on immunophenotypic pattern, majority of the cases were T-B- SCID (39%) followed by T-B+ SCID (28%). MHC class II deficiency accounted for 10.5% of our patient group. A total of 49 patients were molecularly characterized in this study and 32 novel variants were identified in our cohort. The spectrum of genetic defects in our cohort revealed a wide genetic heterogeneity with the major genetic cause being RAG1/2 gene defect (n = 12) followed by IL2RG (n = 9) and JAK3 defects (n = 9). Rare forms of SCID like Purine nucleoside phosphorylase (PNP) deficiency, reticular dysgenesis, DNA-Protein Kinase (DNA-PKcs) deficiency, six cases of MHC class II deficiency and two ZAP70 deficiency were also identified in our cohort. Fourteen percent of the defects still remained uncharacterized despite the application of next generation sequencing. With the exception of MHC class II deficiency and ZAP70 deficiency, all SCID patients had extremely low T cell receptor excision (TRECs) (<18 copies/µL).

9.
J Clin Invest ; 128(7): 3071-3087, 2018 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-29889099

RESUMO

Ikaros/IKZF1 is an essential transcription factor expressed throughout hematopoiesis. IKZF1 is implicated in lymphocyte and myeloid differentiation and negative regulation of cell proliferation. In humans, somatic mutations in IKZF1 have been linked to the development of B cell acute lymphoblastic leukemia (ALL) in children and adults. Recently, heterozygous germline IKZF1 mutations have been identified in patients with a B cell immune deficiency mimicking common variable immunodeficiency. These mutations demonstrated incomplete penetrance and led to haploinsufficiency. Herein, we report 7 unrelated patients with a novel early-onset combined immunodeficiency associated with de novo germline IKZF1 heterozygous mutations affecting amino acid N159 located in the DNA-binding domain of IKZF1. Different bacterial and viral infections were diagnosed, but Pneumocystis jirovecii pneumonia was reported in all patients. One patient developed a T cell ALL. This immunodeficiency was characterized by innate and adaptive immune defects, including low numbers of B cells, neutrophils, eosinophils, and myeloid dendritic cells, as well as T cell and monocyte dysfunctions. Notably, most T cells exhibited a naive phenotype and were unable to evolve into effector memory cells. Functional studies indicated these mutations act as dominant negative. This defect expands the clinical spectrum of human IKZF1-associated diseases from somatic to germline, from haploinsufficient to dominant negative.

11.
Blood ; 130(13): 1553-1564, 2017 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-28778864

RESUMO

NF-κB signaling through its NFKB1-dependent canonical and NFKB2-dependent noncanonical pathways plays distinctive roles in a diverse range of immune processes. Recently, mutations in these 2 genes have been associated with common variable immunodeficiency (CVID). While studying patients with genetically uncharacterized primary immunodeficiencies, we detected 2 novel nonsense gain-of-function (GOF) NFKB2 mutations (E418X and R635X) in 3 patients from 2 families, and a novel missense change (S866R) in another patient. Their immunophenotype was assessed by flow cytometry and protein expression; activation of canonical and noncanonical pathways was examined in peripheral blood mononuclear cells and transfected HEK293T cells through immunoblotting, immunohistochemistry, luciferase activity, real-time polymerase chain reaction, and multiplex assays. The S866R change disrupted a C-terminal NF-κΒ2 critical site affecting protein phosphorylation and nuclear translocation, resulting in CVID with adrenocorticotropic hormone deficiency, growth hormone deficiency, and mild ectodermal dysplasia as previously described. In contrast, the nonsense mutations E418X and R635X observed in 3 patients led to constitutive nuclear localization and activation of both canonical and noncanonical NF-κΒ pathways, resulting in a combined immunodeficiency (CID) without endocrine or ectodermal manifestations. These changes were also found in 2 asymptomatic relatives. Thus, these novel NFKB2 GOF mutations produce a nonfully penetrant CID phenotype through a different pathophysiologic mechanism than previously described for mutations in NFKB2.


Assuntos
Códon sem Sentido , Subunidade p52 de NF-kappa B/genética , Imunodeficiência Combinada Severa/genética , Insuficiência Adrenal/genética , Imunodeficiência de Variável Comum/genética , Displasia Ectodérmica/genética , Hormônio do Crescimento/deficiência , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Fenótipo
12.
Nat Genet ; 49(8): 1192-1201, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28628108

RESUMO

Few monogenic causes for severe manifestations of common allergic diseases have been identified. Through next-generation sequencing on a cohort of patients with severe atopic dermatitis with and without comorbid infections, we found eight individuals, from four families, with novel heterozygous mutations in CARD11, which encodes a scaffolding protein involved in lymphocyte receptor signaling. Disease improved over time in most patients. Transfection of mutant CARD11 expression constructs into T cell lines demonstrated both loss-of-function and dominant-interfering activity upon antigen receptor-induced activation of nuclear factor-κB and mammalian target of rapamycin complex 1 (mTORC1). Patient T cells had similar defects, as well as low production of the cytokine interferon-γ (IFN-γ). The mTORC1 and IFN-γ production defects were partially rescued by supplementation with glutamine, which requires CARD11 for import into T cells. Our findings indicate that a single hypomorphic mutation in CARD11 can cause potentially correctable cellular defects that lead to atopic dermatitis.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Dermatite Atópica/genética , Mutação em Linhagem Germinativa , Guanilato Ciclase/genética , Sistema ASC de Transporte de Aminoácidos/metabolismo , Estudos de Coortes , Análise Mutacional de DNA , Dermatite Atópica/imunologia , Feminino , Genes Dominantes , Glutamina/metabolismo , Humanos , Células Jurkat , Ativação Linfocitária , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Antígenos de Histocompatibilidade Menor/metabolismo , Complexos Multiproteicos/metabolismo , NF-kappa B/metabolismo , Linhagem , Linfócitos T/imunologia , Linfócitos T/metabolismo , Serina-Treonina Quinases TOR/metabolismo
13.
Open Forum Infect Dis ; 4(2): ofx082, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28638843

RESUMO

Idiopathic CD4+ lymphopenia (ICL) predisposes to opportunistic infections (OIs) but can often remain asymptomatic and does not have a strong association with monogenic mutations. Likewise, cryptococcal meningoencephalitis, the most common OI in ICL, is not strongly associated with monogenic mutations. In this study, we describe 2 patients with ICL plus an additional immune defect: one from an E57K genetic mutation in the nuclear factor-κß essential modulator, and the other with acquired autoantibodies to granulocyte-macrophage colony-stimulating factor. Thus, these cases may exemplify a "multi-hit model" in patients with ICL who acquire OIs.

15.
J Exp Med ; 212(10): 1641-62, 2015 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-26304966

RESUMO

Autosomal recessive, complete TYK2 deficiency was previously described in a patient (P1) with intracellular bacterial and viral infections and features of hyper-IgE syndrome (HIES), including atopic dermatitis, high serum IgE levels, and staphylococcal abscesses. We identified seven other TYK2-deficient patients from five families and four different ethnic groups. These patients were homozygous for one of five null mutations, different from that seen in P1. They displayed mycobacterial and/or viral infections, but no HIES. All eight TYK2-deficient patients displayed impaired but not abolished cellular responses to (a) IL-12 and IFN-α/ß, accounting for mycobacterial and viral infections, respectively; (b) IL-23, with normal proportions of circulating IL-17(+) T cells, accounting for their apparent lack of mucocutaneous candidiasis; and (c) IL-10, with no overt clinical consequences, including a lack of inflammatory bowel disease. Cellular responses to IL-21, IL-27, IFN-γ, IL-28/29 (IFN-λ), and leukemia inhibitory factor (LIF) were normal. The leukocytes and fibroblasts of all seven newly identified TYK2-deficient patients, unlike those of P1, responded normally to IL-6, possibly accounting for the lack of HIES in these patients. The expression of exogenous wild-type TYK2 or the silencing of endogenous TYK2 did not rescue IL-6 hyporesponsiveness, suggesting that this phenotype was not a consequence of the TYK2 genotype. The core clinical phenotype of TYK2 deficiency is mycobacterial and/or viral infections, caused by impaired responses to IL-12 and IFN-α/ß. Moreover, impaired IL-6 responses and HIES do not appear to be intrinsic features of TYK2 deficiency in humans.


Assuntos
Síndrome de Job/etiologia , TYK2 Quinase/deficiência , Adolescente , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Interferon gama/metabolismo , Interleucina-10/farmacologia , Interleucina-12/metabolismo , Interleucina-12/farmacologia , Interleucina-23/farmacologia , Interleucina-6/farmacologia , Síndrome de Job/complicações , Síndrome de Job/genética , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Masculino , Mutação , Infecções por Mycobacterium/etiologia , Linfócitos T/metabolismo , Linfócitos T/patologia , TYK2 Quinase/genética , TYK2 Quinase/metabolismo , Viroses/etiologia , Adulto Jovem
16.
J Allergy Clin Immunol ; 136(4): 993-1006.e1, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26162572

RESUMO

BACKGROUND: Follicular helper T (TFH) cells underpin T cell-dependent humoral immunity and the success of most vaccines. TFH cells also contribute to human immune disorders, such as autoimmunity, immunodeficiency, and malignancy. Understanding the molecular requirements for the generation and function of TFH cells will provide strategies for targeting these cells to modulate their behavior in the setting of these immunologic abnormalities. OBJECTIVE: We sought to determine the signaling pathways and cellular interactions required for the development and function of TFH cells in human subjects. METHODS: Human primary immunodeficiencies (PIDs) resulting from monogenic mutations provide a unique opportunity to assess the requirement for particular molecules in regulating human lymphocyte function. Circulating follicular helper T (cTFH) cell subsets, memory B cells, and serum immunoglobulin levels were quantified and functionally assessed in healthy control subjects, as well as in patients with PIDs resulting from mutations in STAT3, STAT1, TYK2, IL21, IL21R, IL10R, IFNGR1/2, IL12RB1, CD40LG, NEMO, ICOS, or BTK. RESULTS: Loss-of-function (LOF) mutations in STAT3, IL10R, CD40LG, NEMO, ICOS, or BTK reduced cTFH cell frequencies. STAT3 and IL21/R LOF and STAT1 gain-of-function mutations skewed cTFH cell differentiation toward a phenotype characterized by overexpression of IFN-γ and programmed death 1. IFN-γ inhibited cTFH cell function in vitro and in vivo, as corroborated by hypergammaglobulinemia in patients with IFNGR1/2, STAT1, and IL12RB1 LOF mutations. CONCLUSION: Specific mutations affect the quantity and quality of cTFH cells, highlighting the need to assess TFH cells in patients by using multiple criteria, including phenotype and function. Furthermore, IFN-γ functions in vivo to restrain TFH cell-induced B-cell differentiation. These findings shed new light on TFH cell biology and the integrated signaling pathways required for their generation, maintenance, and effector function and explain the compromised humoral immunity seen in patients with some PIDs.


Assuntos
Síndromes de Imunodeficiência/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Tirosina Quinase da Agamaglobulinemia , Linfócitos B/imunologia , Ligante de CD40/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Humanos , Quinase I-kappa B/genética , Imunidade Humoral/genética , Síndromes de Imunodeficiência/genética , Memória Imunológica , Proteína Coestimuladora de Linfócitos T Induzíveis/genética , Interferon gama/genética , Interferon gama/metabolismo , Ativação Linfocitária , Mutação/genética , Proteínas Tirosina Quinases/genética , Receptores de Citocinas/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT3/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia
17.
J Clin Immunol ; 35(5): 445-8, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25947952

RESUMO

Severe chronic active Epstein-Barr virus (CAEBV) disease is defined as a severe progressive illness lasting 6 months or longer with infiltration of tissues with EBV-positive lymphocytes, markedly elevated levels of EBV DNA in the blood, and no known immunodeficiency such as HIV. These patients usually have fever, splenomegaly, lymphadenopathy, and may have markedly elevated EBV antibody titers to viral capsid antigen. Although the cause of most cases of severe CAEBV is unknown, one well-documented case was associated with compound heterozygous mutations in PRF1 (perforin 1). Here we report a patient with prolonged severe CAEBV who underwent bone marrow transplant for his disease and subsequently was found to have compound heterozygous mutations in STXBP2 (MUNC18-2) as well as a heterozygous mutation in PRF1 (perforin 1).


Assuntos
Linfócitos B/imunologia , Transplante de Medula Óssea , Infecções por Vírus Epstein-Barr/diagnóstico , Herpesvirus Humano 4/isolamento & purificação , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Proteínas Munc18/genética , Perforina/genética , Adulto , Idade de Início , Linfócitos B/virologia , Biomarcadores/metabolismo , Doença Crônica , Citotoxicidade Imunológica/genética , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/terapia , Grupo com Ancestrais do Continente Europeu , Humanos , Masculino , Mutação/genética , Ativação Viral/genética , Adulto Jovem
18.
Haematologica ; 100(3): 300-7, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25527564

RESUMO

Episodic angioedema with eosinophilia (Gleich syndrome) is a rare disorder characterized by episodes of angioedema and eosinophilia that occur at monthly intervals and resolve spontaneously without therapy. Despite the striking periodicity of this disorder, its similarity to other cyclic hematopoietic disorders with multilineage involvement has not been assessed. To characterize the involvement of cell lineages in the etiology and pathogenesis of episodic angioedema with eosinophilia, four subjects were evaluated by blood counts and other analyses over the course of 1-2 months. Surface marker expression was assessed on T cells by flow cytometry and clonality by polymerase chain reaction. Intracellular cytokine evaluation, bone marrow and skin biopsies were performed during different parts of the cycle. Cycling of multiple cell lineages, including neutrophils, lymphocytes and eosinophils, was observed in the four subjects with the disorder with a periodicity of 25-35 days. An aberrant CD3(-)CD4(+) T-cell population was detected in all four subjects, and T-cell receptor rearrangement studies showed a clonal pattern in three subjects. A peak of type II cytokines was detected in the serum of subjects prior to the onset of symptoms and eosinophil cycling and corresponded to ex-vivo type II cytokines detected intracellularly in CD3(+)CD4(+)CD154(+) T cells. Although the etiology of episodic angioedema with eosinophilia is not yet known, multiple lineages, including lymphocytes, neutrophils and mast cells, are involved and may be related to disease pathogenesis. Whether these cells act directly or promote eosinophilia and eosinophil activation remains to be elucidated. All subjects gave informed consent and were evaluated under an Institutional Review Board-approved protocol (NCT00001406).


Assuntos
Angioedema/diagnóstico , Linhagem da Célula/imunologia , Eosinofilia/diagnóstico , Adulto , Angioedema/complicações , Angioedema/imunologia , Angioedema/patologia , Antígenos CD/imunologia , Medula Óssea/imunologia , Medula Óssea/patologia , Citocinas/imunologia , Eosinofilia/complicações , Eosinofilia/imunologia , Eosinofilia/patologia , Eosinófilos/imunologia , Eosinófilos/patologia , Feminino , Expressão Gênica , Humanos , Imunofenotipagem , Masculino , Mastócitos/imunologia , Mastócitos/patologia , Pessoa de Meia-Idade , Neutrófilos/imunologia , Neutrófilos/patologia , Periodicidade , Pele/imunologia , Pele/patologia , Síndrome , Linfócitos T/imunologia , Linfócitos T/patologia
19.
J Clin Immunol ; 35(1): 11-4, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25352054

RESUMO

Gain of function (GOF) mutation in the p110δ catalytic subunit of the phosphatidylinositol-3-OH kinase (PIK3CD) is the cause of a primary immunodeficiency (PID) characterized by recurrent sinopulmonary infections and lymphoproliferation. We describe a family of two adults and three children with GOF mutation in PIK3CD, all with recurrent sinopulmonary infections and varied infectious and non-infectious complications. The two adults have Primary Sclerosing Cholangitis (PSC) without evidence of Cryptosporidium parvum infection and have required liver transplantation. PSC is a novel phenotype of GOF mutation in PIK3CD.


Assuntos
Colangite Esclerosante/enzimologia , Colangite Esclerosante/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Mutação de Sentido Incorreto , Adulto , Substituição de Aminoácidos , Criança , Colangite Esclerosante/imunologia , Classe I de Fosfatidilinositol 3-Quinases/imunologia , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Feminino , Heterozigoto , Humanos , Síndromes de Imunodeficiência/enzimologia , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Fígado/patologia , Masculino , Linhagem
20.
Front Immunol ; 5: 531, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25404929

RESUMO

BACKGROUND: Primary immunodeficiencies (PIDs) are a diverse group of disorders caused by multiple genetic defects. Obtaining a molecular diagnosis for PID patients using a phenotype-based approach is often complex, expensive, and not always successful. Next-generation sequencing (NGS) methods offer an unbiased genotype-based approach, which can facilitate molecular diagnostics. OBJECTIVE: To develop an efficient NGS method to identify variants in PID-related genes. METHODS: We performed HaloPlex custom target enrichment and NGS using the Ion Torrent PGM to screen 173 genes in 11 healthy controls, 13 PID patients previously evaluated with either an identified mutation or SNP, and 120 patients with undiagnosed PIDs. Sensitivity and specificity were determined by comparing NGS and Sanger sequencing results for 33 patients. Run metrics and coverage analyses were done to identify systematic deficiencies. RESULTS: A molecular diagnosis was identified for 18 of 120 patients who previously lacked a genetic diagnosis, including 9 who had atypical presentations and extensive previous genetic and functional studies. Our NGS method detected variants with 98.1% sensitivity and >99.9% specificity. Uniformity was variable (72-89%), and we were not able to reliably sequence 45 regions (45/2455 or 1.8% of total regions) due to low (<20) average read depth or <90% region coverage; thus, we optimized probe hybridization conditions to improve read-depth and coverage for future analyses, and established criteria to help identify true positives. CONCLUSION: While NGS methods are not as sensitive as Sanger sequencing for individual genes, targeted NGS is a cost-effective, first-line genetic test for the evaluation of patients with PIDs. This approach decreases time to diagnosis, increases diagnostic rate, and provides insight into the genotype-phenotype correlation of PIDs in a cost-effective way.

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