Identification of an X chromosome microdeletion in a patient with recurrent fever and bacteremia
The patient had an unremarkable medical history, with the exception of atopic dermatitis and asthma during childhood, both of which improved with age. In 2018, the patient experienced an episode of aseptic meningitis. In 2019, multiple events of gross hematuria and proteinuria occurred, followed by recurrent fever up to over 40℃ and confirmation of bacteremia (Fig. 1A) caused by atypical bacteria usually not found in a clinical setting. In addition, CT scans revealed waxing and waning ground-glass opacities (GGO) in both lungs. We also found renal involvement (hematuria and proteinuria), a history of asthma, and positivity for antineutrophil cytoplasmic antibodies (p-ANCA) to MPO, raising a suspicion of ANCA-associated vasculitis. Subsequently, in the second half of 2020, the patient suffered from persistent diarrhea and hematochezia, and atypical ulcerative colitis was suspected with chronic colitis diagnosed by colonoscopy. Meanwhile, there was no significant family history (Fig. 1B).
To exclude a genetic cause underlying these clinical manifestations, we performed trio whole-exome sequencing (WES). However, no clinically significant sequence variants were detected via trio WES. Additionally, CCR-CNV, an in-house CNV prediction tool, indicated a heterozygous deletion of the X chromosome (data not shown) 9. Inspection of the region by integrative genomics viewer (IGV) revealed reduced depth (Supplementary Fig. S1). To confirm this deletion, we conducted chromosomal microarray analysis (CMA), which revealed a deletion of approximately 4,480 kb on the X chromosome (arr[GRCh37] Xp11.23p11.22(48225025_52705915)×1; Fig. 1C). This deletion was also identified in the patient’s mother (Fig. 1D). This region encompasses 19 genes reported in Online Mendelian Inheritance in Man (OMIM, https://www.omim.org/), including FOXP3 and WAS (Fig. 1E). Considering that this was a heterozygous X chromosome deletion in a female patient, we performed an X chromosome inactivation (XCI) assay to assess skewing, and found a skewed XCI pattern in both the patient and her mother (Fig. 1F), indicating inactivation of the 22-repeat allele in the patient (> 85:15). Notably, the patient exhibited incomplete skewing (approximately 85%), whereas the mother showed complete skewing (100%). To the best of our knowledge, this is the first report of a female patient presenting with a Xp11.23-p11.22 microdeletion with a skewed XCI pattern, in which the symptomatic mutated alleles are activated preferentially.
Immunological features of the patient
In order to determine whether the defect of the immune cells of patient lead to phenotype, we applied immune cell analysis from patient with Xp microdeletion. Analysis of the patient’s peripheral blood mononuclear cells (PBMCs) revealed a significant increase in the Th1, Th2, and Th17 cell populations (Fig. 2A–C). Surface expression of activation markers such as CD25, CD44, and CD69, as well as the memory marker (CD45RO), did not differ between patient and healthy donor (HD) CD4 T cells after in vitro stimulation (Fig. 2D). Serum cytokines, including IL-17A, IFN-γ, TNF, IL-10, IL-6, IL-4, and IL-2 levels from the patient and HD were measured using a Cytokine Bead Array. The results revealed an increase in proinflammatory cytokine levels in the patient’s serum (Fig. 2E). In addition, the effector/memory CD4 T cell population was higher in the patient than in the HD (Fig. 2F). Our case is heterozygous for the deleted Xp11.23-p11.22 chromosome region, but various genes including FOXP3 and WAS genes, which are important for Treg cell function10,11. It is somewhat similar to, but different from, previously reported cases of WAS and FOXP3 deficiency associated with Xp11.23 11,12. The proportion of Treg cells in the patient was no different from that in the HD (Fig. 3A), while the level of GITR was lower than that in the HD (Fig. 3B). Remarkably, there was a notable reduction in the levels of the Treg cell function-related cytokines TGF-β1 and IL-10 levels in the patient (Fig. 3C, D). In addition, induction of in vitro-induced Treg (iTreg) cell differentiation was less efficient in the patient than in the HD (Fig. 3E). The observed iTreg differentiation differences in the same type of cells under in vitro conditions are presumed to be due to an intrinsic predisposition driven by the combined effect of Xp microdeletion and skewed inactivation within CD4 T cells. These dysfunctional Treg cells may render the patient susceptible to the development of immune-mediated inflammatory diseases.
scRNA-Seq of CD4 T cells from the patient and HD
Given the importance of CD4 T cells for disease pathogenesis, we next sequenced CD4 T cell RNA using the 10x Genomics Gem Code Chromium platform to explore transcriptomic differences between the patient and HD. After quality control, we obtained expression profiles for 9,886 and 7,207 CD4 T cells from the HD and patient, respectively. Dimension reduction by Uniform Manifold Approximation and Projection (UMAP) identified 10 clusters: three clusters of CD4 naïve T cells and seven clusters of CD4 effector/memory T cells, each with unique signature genes (Fig. 4A). To identify the specific CD4 T cell types associated with symptoms, we integrated data from the HD and patient (Fig. 4B). We next identified differentially expressed RNA markers in each cluster, and found that each exhibited distinct molecular patterns and biomarkers (Fig. 4C and Supplementary Fig. S2).
To identify differences in cell composition across samples, the clusters were examined separately (Supplementary Fig. S3A), and the percentage of cells within the 10 major cell clusters was calculated for each individual (Supplementary Fig. S3B). Most cell types exhibited overlap between the patient and HD. Notably, the patient had a much higher percentage of cytotoxic CD4 T cells (CD4 CTLs) than the HD (Supplementary Fig. S3B). In general, cytotoxic CD4 T cell (CD4 CTL) numbers are low under normal conditions 13,14. In humans, CD4 CTL numbers increase in response to chronic viral infection 15–17, anti-tumor responses 18,19 and several autoimmune diseases 20–22. The CD4 CTL cluster was characterized by high expression of genes associated with the cytotoxic functions of CD8+ T cells and natural killer (NK) cells; these included NKG7, GZMA, CST7, GNLY, PRF1, FGFBP2, PLEK, GZMB, KLRG1, GZMH, CTSW, ZNF683, SPON2, TBX21, and EFHD2. These CD4 CTL signature genes were overall higher in cluster of the patient than healthy donor (Fig. 4D, F). Although expression of Treg cell function-related proteins was reduced (Fig. 3), scRNA-seq did not reveal a change in expression of Treg-related genes (Fig. 4E, G). Furthermore, the proportion of CD4 TEMs was considerably lower in the patient than in the HD (Supplementary Fig. S3B), while expression of interferon (IFN)-stimulated genes (ISGs) including IFI44L, ISG15, MX1, IRF7, and IRF1 was upregulated in the CD4 TEM (High ISG) cluster from the patient (Supplementary S3C, S3D). These findings suggest that immunological abnormalities in the patient from changes in gene expression of CD4 T cells induced by Xp microdeletion.
Marked clonal expansion of CD4 CTLs from the patient
To further determine the characteristics of CD4 T cells in the patient, TCR sequencing was performed using the 10× Genomics V(D)J-enriched library. CD4 T cell TCR diversity was assessed in the patient and HD. Definition of clonotypes was based on CDR3 sequencing of both TCR alpha and beta chains using the Cell Ranger analysis pipeline. Clonotypic analysis of the TCR was conducted using the scRepertoire package 23. We calculated the percentage of each unique T cell clonotype; most CD4 T cells from the HD contained unique TCRs, while those from the patient showed a reduced percentage of unique clonotypes (Fig. 5A). Next, we analyzed the length of the CDR3 nucleotides in the TCR alpha and beta chains. The distribution of CDR3 length in both TRA and TRB from the HD and patient were similar, but not completely identical, to the normal distribution. More clonotypes were found at 33, 36, 39, 42, 45, 48 nucleotides from healthy donor (Fig. 5B). These findings also indicate that the TCR repertoire of patient is less diverse than that of the HD. Moreover, the relative abundance of highly expanded clonotypes was higher in the patient (Fig. 5C). From the TCR repertoire analysis of these paired TCRαβ sequences, we represented the top 10 TCR clonotypes for each sample. In the case of HD, there were few overlapping clones in the top 10; however, the proportion of expanded clones in the top 10 was higher in the patient (Fig. 5D). No TCR clonotype was shared between the patient and HD (Fig. 5E). Most of these expanded TCR clonotypes were found in the CD4 TEMRA cluster (CD4 CTL) (Fig. 5F–H). In the CD4 TEMRA cluster (CD4 CTL), in which expanded clones comprised the highest proportion, there were a total of 258 large clone types, ranging from 20–100; of these, 256 expanded clones were found in the patient (Fig. 5F). Public databases were mined to assess the known antigen specificity of CDR3 sequences. We analyzed the top five expanded clonotypes in CD4 CTLs using TCR databases VDJdb 24 and McPAS-TCR 25; however, none of the clonotypes were found. The CD4 CTLs are known to be highly heterogenous across patients26; therefore, it is not entirely surprising that the expanded clonotypes in the patient did not match those deposited in TCR databases.