Germline gain-of-function mutation of STAT1 rescued by somatic mosaicism in immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like disorder
Authors: Jeong Seok Lee, Yohan An, Christopher J. Yoon, Jeong Yeon Kim, Kyung Hwan Kim, Alexandra F. Freeman, Jae-Joon Yim, Eui-Cheol Shin, Steven M. Holland, Eun Young Lee, Young Seok Ju
Journal: Journal of Allergy and Clinical Immunology (2019)

Gain-of-function (GoF) mutations of signal transducer and activator of transcription 1 (STAT1) cause broad immune dysfunction ranging from simple immunodeficiency to complex immune disorders.1Uzel G. Sampaio E.P. Lawrence M.G. Hsu A.P. Hackett M. Dorsey M.J. et al.Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.J Allergy Clin Immunol. 2013; 131: 1611-1623Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar These mutations are frequently enriched in the coiled-coil domain or DNA-binding domain of STAT1 (Fig 1, A).2Toubiana J. Okada S. Hiller J. Oleastro M. Lagos Gomez M. Aldave Becerra J.C. et al.Heterozygous STAT1 gain-of-function mutations underlie an unexpectedly broad clinical phenotype.Blood. 2016; 127: 3154-3164Crossref PubMed Scopus (328) Google Scholar,3Leiding J.W. Okada S. Hagin D. Abinun M. Shcherbina A. Balashov D.N. et al.Hematopoietic stem cell transplantation in patients with gain-of-function signal transducer and activator of transcription 1 mutations.J Allergy Clin Immunol. 2018; 141: 704-717.e5Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar STAT1 GoF mutations lead to the uncontrolled phosphorylation of STAT1, which disrupts STAT1-dependent clonal expansion and memory formation of CD8+ T cells.4Quigley M. Huang X. Yang Y. STAT1 signaling in CD8 T cells is required for their clonal expansion and memory formation following viral infection in vivo.J Immunol. 2008; 180: 2158-2164Crossref PubMed Scopus (45) Google Scholar,5Sampaio E.P. Hsu A.P. Pechacek J. Bax H.I. Dias D.L. Paulson M.L. et al.Signal transducer and activator of transcription 1 (STAT1) gain-of-function mutations and disseminated coccidioidomycosis and histoplasmosis.J Allergy Clin Immunol. 2013; 131: 1624-1634Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar The most frequent clinical phenotype induced by STAT1 GoF mutations is simple immunodeficiency, for example, chronic mucocutaneous candidiasis (Fig 1, B).6van de Veerdonk F.L. Plantinga T.S. Hoischen A. Smeekens S.P. Joosten L.A. Gilissen C. et al.STAT1 mutations in autosomal dominant chronic mucocutaneous candidiasis.N Engl J Med. 2011; 365: 54-61Crossref PubMed Scopus (501) Google Scholar In a very rare circumstance, GoF STAT1 mutations result in aggressive immune phenotypes, similar to immune dysregulation-polyendocrinopathy-enteropathy-X-linked (IPEX) syndrome caused by FOXP3 loss-of-function mutations. In the IPEX-like disorder (IPEX-LD),1Uzel G. Sampaio E.P. Lawrence M.G. Hsu A.P. Hackett M. Dorsey M.J. et al.Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.J Allergy Clin Immunol. 2013; 131: 1611-1623Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar severe immunodeficiency and autoimmune phenotypes are combined (Fig 1, B). However, the molecular mechanisms underlying the broad clinical phenotypes of STAT1 GoF mutations remain elusive (see Table E1 in this article's Online Repository at www.jacionline.org). Here we conducted a comprehensive whole-genome analysis of a patient with IPEX-LD. Medical history and laboratory information were obtained through a medical chart review (see Table E2 in this article's Online Repository at www.jacionline.org). Over her lifetime, she showed multiple immunodeficient and autoimmune phenotypes (Fig 1, C).1Uzel G. Sampaio E.P. Lawrence M.G. Hsu A.P. Hackett M. Dorsey M.J. et al.Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.J Allergy Clin Immunol. 2013; 131: 1611-1623Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar The patient exhibited lower natural killer cell cytotoxicity and TH17 cell count than healthy controls as previously reported (Fig E1; see this article's Methods section in the Online Repository at www.jacionline.org).1Uzel G. Sampaio E.P. Lawrence M.G. Hsu A.P. Hackett M. Dorsey M.J. et al.Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.J Allergy Clin Immunol. 2013; 131: 1611-1623Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar,E12 Serum IgE level was within normal range unlike in FOXP3-mutated IPEX syndrome. The patient harbored a de novo STAT1 T385M GoF mutation (chr2:191,851,647 G>A substitution) (Fig 1, D).1Uzel G. Sampaio E.P. Lawrence M.G. Hsu A.P. Hackett M. Dorsey M.J. et al.Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.J Allergy Clin Immunol. 2013; 131: 1611-1623Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar The mutation was not present in the genomes of either the parents or her sister. The variant allele fraction of the mutation was approximately 50%, consistent with heterozygous mutations present in all the cells of the proband. The variant was phased to the reference allele of the maternally inherited heterozygous polymorphism (chr2:191,851,997 T>C substitution rs17817076), suggesting that the mutation was acquired through the maternal germline lineage (Fig 1, D). To identify additional genomic alterations that conferred the patient's extreme IPEX-LD manifestation, we systematically analyzed her entire genome sequences targeting 4 variant classes (see Tables E3-E5 in this article's Online Repository at www.jacionline.org). However, given the genomic positions of those alterations, none of (1) germline substitutions/indels and (2) de novo mutations seemed to be associated with the disease phenotypes and (3) structural variations (rearrangements and retrotransposon mobilization) were not found. Finally, we explored the impact of (4) somatic mosaicism. Of the 2474 somatic mosaicism candidates from whole-genome sequencing (2043 base substitutions and 431 indels; see Table E6 in this article's Online Repository at www.jacionline.org), 15 (0.6 %) were located in protein-coding regions. The most interesting candidate was a frameshift T insertion (chr2:191,851,636 C>CT) in the STAT1 coding sequence, 11 bp from the de novo T385M GoF mutation (Fig 1, D). Consistent with somatic mosaicism, this variant was not found in other family members and other unmatched normal populations (0 of 15,708 whole-genome sequences from gnomAD (v2.1.1) and 0 of 125,748 whole-exome sequences from ExAC consortium),7Lek M. Karczewski K.J. Minikel E.V. Samocha K.E. Banks E. Fennell T. et al.Analysis of protein-coding genetic variation in 60,706 humans.Nature. 2016; 536: 285-291Crossref PubMed Scopus (6587) Google Scholar but was present in 8.1% of the patient's blood cells (3 supporting reads of the 37 reads from the maternal allele; 95% CI, 1.7%-23.7%, Poisson exact test). Because the T insertion was phased to the T385M mutation (Fig 1, D), the somatic mutation likely abrogates the impact of the de novo GoF STAT1 mutation in the fraction of patient's blood cells. To understand the quantitative impact and the cell-type distribution of the secondary STAT1 mutation, we confirmed the burden of mutations using ultra-high depth of amplicon sequencing (>1,000,000-fold) from different cell types including buccal swabs and specific blood cells. The mutation was present at variable fractions in different cell types, that is, buccal epithelial cells (∼0%; 217 of 645,926), CD8+ (15.8%; 93,048 of 589,788; 95% CI, 15.7%-15.9%), CD4+ (7.0%; 47,935 of 682,072; 95% CI, 6.96%-7.09%), CD19+ (2.5%; 17,535 of 692,530; 95% CI, 2.49%-2.57%), and CD14+ (0.6%; 5,348 of 874,933; 95% CI, 0.59%-0.62%) cells (Fig 1, E). We further looked for other somatic mutations in CD8+ cells using 70-fold whole-genome sequencing, but no remarkable mutations other than the STAT1 T insertion were identified. Our findings suggest that at least 2 different sets of blood cells were present in the patient with IPEX-LD: (1) single T385M STAT1-mutant cells and (2) double-mutant (T385M and T insertion) STAT1-mutant blood cells (Fig 1, D, and Fig 2). In theory, the single-mutant cells will be nonfunctional due to the impaired STAT1 signaling, leading to immunodeficiency. In contrast, the double-mutant T cells with restored STAT1 signaling could be more reactive than single-mutant cells to antigens because the T385M GoF STAT1 mutation is not expressed. Because normal STAT1 signaling confers cytotoxic activity, we believe that some, if not all, of the double STAT1-mutant CD8+ T cells may contribute to the multiple extreme autoimmune phenotypes manifested in the patient with IPEX-LD. Experimental studies suggest that the double-mutant cells are functionally distinct from the single-mutant cells. U3A cells with frameshift T insertion in combination with STAT1 T385M GoF mutation exhibited a full inactivation of STAT1 T385M mutant alleles in response to IFN-γ (Fig 1, F). In addition, Jurkat T cells transfected with STAT1 harboring both the T385M and the T insertion showed the higher cellular proliferation rate after IFN-γ stimulation than did cells transfected with only the T385M mutation (P = .0066), which is close to the rate observed in the wild-type STAT1 cells (Fig 1, G). The somatic mutation was not identified in the cells of endodermal origin (buccal cells), but was present in mesodermal blood cells. This suggests that the somatic mosaicism likely emerged after the formation of the 3 germ layers in embryogenesis but earlier than the cell fate determination of pluripotent hematopoietic stem cells (Fig 2). The reason underlying the higher frequency of double-mutant clones in CD8+ T cells is unclear. In theory, it can be caused by (1) more frequent fate determination into CD8+ T cells for double STAT1-mutant hematopoietic stem cells, (2) moderate clonal expansion of double STAT1-mutant CD8+ T cells by restoration of STAT1-dependent inhibition of CD8+ T-cell proliferation in periphery, or both.8Gil M.P. Ploquin M.J. Watford W.T. Lee S.H. Kim K. Wang X. et al.Regulating type 1 IFN effects in CD8 T cells during viral infections: changing STAT4 and STAT1 expression for function.Blood. 2012; 120: 3718-3728Crossref PubMed Scopus (59) Google Scholar Of note, we found a few TCR clonotypes of minor contributions (<3%) from bulk RNA-seq of PBMCs (see Table E7 in this article's Online Repository at www.jacionline.org) although their STAT1 genotypes are unclear. The functional importance of early somatic mosaicisms is emerging.9Ju Y.S. Martincorena I. Gerstung M. Petljak M. Alexandrov L.B. Rahbari R. et al.Somatic mutations reveal asymmetric cellular dynamics in the early human embryo.Nature. 2017; 543: 714-718Crossref PubMed Scopus (155) Google Scholar Given the mutation rate in early embryogenesis (∼3 base substitutions per cell per cell doubling), a 16-cell embryo will collectively acquire approximately 90 somatic base substitutions, which will be present in most cell types of the individual. In addition, much later mutations can also affect a substantial proportion of specific cell types if they were enriched in founder cells of a specific cell type. However, because of technical limitations, studies targeting somatic mosaicisms are lacking. Our study demonstrates that comprehensive whole-genome analyses are necessary to understand the frequency and the functional roles of somatic mosaicisms in the phenotypic diversity of rare human diseases. We thank Professors Sei Won Yang, Choong-Ho Shin, and Young Ah Lee of Seoul National University Children's Hospital for the care of this patient for a long time. Most of all, we give special thanks to the dedicated efforts of the patient and her family who have made this study possible. We thank Ji Yong Choi for her technical assistance. Download .docx (.02 MB) Help with docx files Online Repository text Download .pdf (.02 MB) Help with pdf files Fig E1 Download .docx (.02 MB) Help with docx files Table E1 Download .docx (.02 MB) Help with docx files Table E2 Download .xlsx (.01 MB) Help with xlsx files Table E3 Download .xlsx (.01 MB) Help with xlsx files Table E4 Download .xlsx (.01 MB) Help with xlsx files Table E5 Download .xlsx (.16 MB) Help with xlsx files Table E6 Download .xlsx (.04 MB) Help with xlsx files Table E7