CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response
Haapaniemi E, Botla S, Persson J, Schmierer B, Taipale J. Nat Med, 2018. 24(7): p.927-930
In this publication we report that genome editing by CRISPR–Cas9 induces a p53-mediated DNA damage response and cell cycle arrest in immortalized human retinal pigment epithelial cells, leading to a selection against cells with a functional p53 pathway. Inhibition of p53 prevents the damage response and increases the rate of homologous recombination from a donor template. These results suggest that p53 inhibition may improve the efficiency of genome editing of untransformed cells and that p53 function should be monitored when developing cell-based therapies utilizing CRISPR–Cas9.
All publications in chronological order
Dominant TOM1 mutation associated with multi-organ autoimmune disease
Keskitalo S*, Haapaniemi E*, Glumoff V, Liu X, Fogarty C, Lehtinen V, Rajala H, Chian-Cher S, Mustjoki S, Kovanen P, Lohi J, Bryceson Y, Seppänen M, Kere J, Heiskanen K, Varjosalo M. Genomic Medicine in press.
Mutations in several proteins functioning as endolysosomal components cause monogenic autoimmune diseases, of which pathogenesis is linked to increased endoplasmic reticulum stress, inefficient autophagy, and defective recycling of immune receptors.
We report here a heterozygous TOM1 p.G307D missense mutation, detected by whole-exome sequencing, in two related patients presenting with early-onset autoimmunity, antibody deficiency, and features of combined immunodeficiency.
The index patient suffered from recurrent respiratory tract infections and oligoarthritis since early teens, and later developed persistent low-copy EBV-viremia, as well as an antibody deficiency. Her infant son developed hypogammaglobulinemia, autoimmune enteropathy, interstitial lung disease, profound growth failure, and treatment-resistant psoriasis vulgaris.
Consistent with previous knowledge on TOM1 protein function, we detected impaired autophagy and enhanced susceptibility to apoptosis in patient-derived cells. In addition, we noted diminished STAT and ERK1/2 signaling in patient fibroblasts, as well as poor IFN-γ and IL-17 secretion in T cells. The mutant TOM1 failed to interact with TOLLIP, a protein required for IL-1 recycling, PAMP signaling and autophagosome maturation, further strengthening the link between the candidate mutation and patient pathophysiology.
In sum, we report here an identification of a novel gene, TOM1, associating with early-onset autoimmunity, antibody deficiency, and features of combined immunodeficiency. Other patient cases from unrelated families are needed to firmly establish a causal relationship between the genotype and the phenotype.
Heterozygous activating mutation in RAC2 causes infantile-onset combined immunodeficiency with susceptibility to viral infections
Sharapova SO, Haapaniemi E, Sakovich IS, Kostyuchenko LV, Donkó A, Dulau-Florea A, Malko O, Bondarenko AV, Stegantseva MV, Leto TL, Uygun V, Karasu GT, Holland SM, Hsu AP, Aleinikova OV. J. Clin Immunol. 2019 May 6 [Epub ahead of print].
Here we describe a 10-year-old girl with combined immunodeficiency presenting as recurring chest infections, lung disease and herpetic skin infections. The patient experienced two hematopoietic stem cell transplantations and despite full chimerism, she developed bone marrow aplasia due to adenovirus infection and died at post-transplant day 86.
Immunologic investigation revealed low numbers of TRECs/KRECs, a severe reduction of memory B cells, absence of isohemagglutinins, and low IgG levels. Whole exome sequencing (WES) identified a novel heterozygous mutation in RAC2(c.275A > C, p.N92 T). Flow cytometric investigation of neutrophil migration demonstrated an absence of chemotaxis to fMLP. Cell lines transfected with RAC2 [N92 T] displayed characteristics of active GTP-bound RAC2 including enhanced reactive oxygen species (ROS) production both at rest and in response to PMA. Our findings broaden the clinical picture of RAC2 dysfunction, showing that some individuals can present with a combined immunodeficiency later in childhood rather than a congenital neutrophil disease.
Characterization of novel TMEM173 mutation with additive IFIH1 risk allele
Keskitalo S, Haapaniemi E, Einarsdottir E, Rajamaki K, Heikkila H, Ilander M, Poyhonen M, Morgunova E, Hokynar K, Lagstrom S, Kivirikko S, Mustjoki S, Eklund K, Saarela J, Kere J, Seppanen M, Ranki A, Hannula-Jouppi K, Varjosalo M.
TMEM173 encodes for STING that is a transmembrane protein activated by pathogen or self-derived cytosolic nucleic acids causing its translocation from ER to Golgi, and further to vesicles. Monogenic STING gain-of-function mutations cause early-onset type I interferonopathy, with disease presentation ranging from fatal vasculopathy to mild chilblain lupus. Molecular mechanisms causing the poor phenotypegenotype correlation are presently unclear.
Here we report a novel gain-of-function G207E STING mutation causing a distinct phenotype with alopecia, photosensitivity, thyroid dysfunction, and STING-associated vasculopathy with onset in infancy (SAVI) -features; livedo reticularis, nasal septum perforation, facial erythema, bacterial infections and skin vasculitis. Single residue polymorphisms in TMEM173 and an IFIH1 T946 risk allele modify disease presentation in the affected multigeneration family, explaining the varying clinical phenotypes. The G207E mutation causes constitutive activation of inflammation-related pathways in HEK cells, as well as aberrant interferon signature and inflammasome activation in patient PBMCs. Protein-protein interactions further propose impaired cellular trafficking of G207E mutant STING.
These findings reveal the molecular landscape of STING and highlight the complex additive effects on the phenotype.
Novel LRBA Mutation and Possible Germinal Mosaicism in a Slavic Family
Sharapova S, Haapaniemi E, Sakovich I, Rojas J, Gámez-Díaz L, Mareika Y, Guryanova I, Migas A, Mikhaleuskaya T, Grimbacher B, Aleinikova O. J Clin Immunol. 2018 38(4): p.471-474
Here, we report the clinical, genetic, and immunological data from two siblings in a Belarusian family with a homozygous, truncating LRBA mutation (c.2762G>C, p.Ser921Stop). Patient 1 (P1), male, was born at term to non-consanguineous parents.
Damaging heterozygous mutations in NFKB1 lead to diverse immunologic phenotypes
Kaustio M*, Haapaniemi E*, Nurkkala H*, Helminen M, Park G, Syrjänen J, Einarsdottir E, Sahu B, Kilpinen S, Rounioja S, Fogarty C, Glumoff V, Kulmala P, Katayama S, Tamene F, Trotta L, Morungova E, Krjutskov K, Martelius T, Mustjoki S, Taipale J, Saarela J, Kere J, Varjosalo M, Seppänen M. Journal of Allergy and Clinical Immunology 2017. 40(3): p.782-796
The nuclear factor κ light-chain enhancer of activated B cells (NF-κB) signaling pathway is a key regulator of immune responses. Accordingly, mutations in several NF-κB pathway genes cause immunodeficiency.
We sought to identify the cause of disease in 3 unrelated Finnish kindreds with variable symptoms of immunodeficiency and autoinflammation.
We applied genetic linkage analysis and next-generation sequencing and functional analyses of NFKB1 and its mutated alleles.
In all affected subjects we detected novel heterozygous variants in NFKB1, encoding for p50/p105. Symptoms in variant carriers differed depending on the mutation. Patients harboring a p.I553M variant presented with antibody deficiency, infection susceptibility, and multiorgan autoimmunity. Patients with a p.H67R substitution had antibody deficiency and experienced autoinflammatory episodes, including aphthae, gastrointestinal disease, febrile attacks, and small-vessel vasculitis characteristic of Behçet disease. Patients with a p.R157X stop-gain experienced hyperinflammatory responses to surgery and showed enhanced inflammasome activation. In functional analyses the p.R157X variant caused proteasome-dependent degradation of both the truncated and wild-type proteins, leading to a dramatic loss of p50/p105. The p.H67R variant reduced nuclear entry of p50 and showed decreased transcriptional activity in luciferase reporter assays. The p.I553M mutation in turn showed no change in p50 function but exhibited reduced p105 phosphorylation and stability. Affinity purification mass spectrometry also demonstrated that both missense variants led to altered protein-protein interactions.
Our findings broaden the scope of phenotypes caused by mutations in NFKB1 and suggest that a subset of autoinflammatory diseases, such as Behçet disease, can be caused by rare monogenic variants in genes of the NF-κB pathway.
Combined immunodeficiency and hypoglycemia associated with mutations in hypoxia up-regulated 1
Haapaniemi E, Fogarty C, Katayama S, Vihinen H, Keskitalo S, Ilander M, Krjutškov K, Mustjoki S, Lehto M, Hautala T, Jokitalo E, Velagapudi V, Varjosalo M, Seppänen M and Kere J. Journal of Allergy and Clinical Immunology 2017 Apr;139(4): p.1391-1393
Inherited DOCK2 Deficiency in Patients with Early-Onset Invasive Infections
Dobbs K, Dominguez‐Conde C Zhang SY, Parolini S, Audry M, Chou J, Haapaniemi E, Keles S, Bilic I, Okada S, Massaad MJ, Rounioja S, Alwahadneh AM, Serwas NK, Capuder K, Ciftci E, Felgentreff K, Ohsumi T, Pedergnana V, Boisson B, Haskoloğlu S, Ensari A, Schuster M, Moretta A, Itan Y, Patrizi O, Rozenberg F, Lebon P, Saarela J, Knip M, Petrovski S, Goldstein DB, Parrott RE, Savas B, Schambach A, Tabellini G, Bock C, Chatila T, Comeau AM, Geha RS, Abel L, Buckley RH, Ikincioğullari A, Al‐Herz W, Helminen M, Doğu F, Casanova JL, Boztuğ K, Notarangelo LD. N Engl J Med 2015; 372:2409-2422
Combined immunodeficiencies are marked by inborn errors of T-cell immunity in which the T cells that are present are quantitatively or functionally deficient. Impaired humoral immunity is also common. Patients have severe infections, autoimmunity, or both. The specific molecular, cellular, and clinical features of many types of combined immunodeficiencies remain unknown.
We performed genetic and cellular immunologic studies involving five unrelated children with early-onset invasive bacterial and viral infections, lymphopenia, and defective T-cell, B-cell, and natural killer (NK)–cell responses. Two patients died early in childhood; after allogeneic hematopoietic stem-cell transplantation, the other three had normalization of T-cell function and clinical improvement.
We identified biallelic mutations in the dedicator of cytokinesis 2 gene (DOCK2) in these five patients. RAC1 activation was impaired in the T cells. Chemokine-induced migration and actin polymerization were defective in the T cells, B cells, and NK cells. NK-cell degranulation was also affected. Interferon-α and interferon-λ production by peripheral-blood mononuclear cells was diminished after viral infection. Moreover, in DOCK2-deficient fibroblasts, viral replication was increased and virus-induced cell death was enhanced; these conditions were normalized by treatment with interferon alfa-2b or after expression of wild-type DOCK2.
Autosomal recessive DOCK2 deficiency is a new mendelian disorder with pleiotropic defects of hematopoietic and nonhematopoietic immunity. Children with clinical features of combined immunodeficiencies, especially with early-onset, invasive infections, may have this condition. (Supported by the National Institutes of Health and others.)
Autoimmunity, hypogammaglobulinemia, lymphoproliferation, and mycobacterial disease in patients with activating mutations in STAT3
Haapaniemi E, Kaustio M, Rajala H, van Adrichem A, Kainulainen L, Glumoff V, Doffinger R, Kuusanmäki H, Heiskanen-Kosma T, Trotta L, Chiang S, Kulmala P, Eldfors S, Katainen R, Siitonen S, Karjalainen-Lindsberg M, Kovanen P, Otonkoski T, Porkka K, Heiskanen K, Hänninen A, Bryceson Y, Uusitalo-Seppälä R, Saarela J, Seppänen M, Mustjoki S, Kere J. Blood, 2015. 125(4): p. 639-48.
The signal transducer and activator of transcription (STAT) family of transcription factors orchestrate hematopoietic cell differentiation. Recently, mutations in STAT1, STAT5B, and STAT3 have been linked to development of immunodysregulation polyendocrinopathy enteropathy X-linked–like syndrome.
Here, we immunologically characterized 3 patients with de novo activating mutations in the DNA binding or dimerization domains of STAT3 (p.K392R, p.M394T, and p.K658N, respectively). The patients displayed multiorgan autoimmunity, lymphoproliferation, and delayed-onset mycobacterial disease. Immunologically, we noted hypogammaglobulinemia with terminal B-cell maturation arrest, dendritic cell deficiency, peripheral eosinopenia, increased double-negative (CD4−CD8−) T cells, and decreased natural killer, T helper 17, and regulatory T-cell numbers. Notably, the patient harboring the K392R mutation developed T-cell large granular lymphocytic leukemia at age 14 years.
Our results broaden the spectrum of phenotypes caused by activating STAT3 mutations, highlight the role of STAT3 in the development and differentiation of multiple immune cell lineages, and strengthen the link between the STAT family of transcription factors and autoimmunity.
Activating germline mutations in STAT3 cause early-onset multi-organ autoimmune disease
Flanagan S.E*, Haapaniemi E*, Russel M*, Caswell R, Lango Allen H, De Franco E, McDonald T, Rajala H, Ramelius A, Barton J, Heiskanen K, Heiskanen-Kosma T, Kajosaari M, Murphy N, Milenkovic T, Seppänen M, Lenmark Å, Mustjoki S, Otonkoski T, Kere J, Morgan N, Ellard S, Hattersley A. Nat Genet, 2014. 46(8): p. 812-4
Monogenic causes of autoimmunity provide key insights into the complex regulation of the immune system. We report a new monogenic cause of autoimmunity resulting from de novo germline activating STAT3 mutations in five individuals with a spectrum of early-onset autoimmune disease, including type 1 diabetes. These findings emphasize the critical role of STAT3 in autoimmune disease and contrast with the germline inactivating STAT3 mutations that result in hyper IgE syndrome.
* Equal contribution