A novel homozygous RHOH variant associated with T cell dysfunction and recurrent opportunistic infections

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A deficiency in RHOH has been linked to epidermodysplasia verruciformis, lung disease, Burkitt lymphoma and T cell defects. Here, we report a novel germline homozygous RHOH c.245G > A (p.Cys82Tyr) variant in a 21-year-old male suffering from recurrent, invasive, opportunistic infections affecting the lungs, eyes, and brain. His sister also succumbed to a lung infection during early adulthood. The patient exhibited a persistent decrease in CD4 + T, B, and NK cell counts, and hypoimmunoglobulinemia. Despite normal RHOH mRNA levels in his family, the patient’s T cell showed impaired activation upon in vitro TCR stimulation. In Jurkat T cells transduced with RHOH C82Y , a similar reduction in CD69 activation marker up-regulation was observed. However, ectopic expression of the C82Y variant did not exhibit a negative dominance over wild type RHOH. Furthermore, the C82Y variant showed reduced RHOH protein expression and impaired interaction with the TCR signaling molecule ZAP70. Together, these data suggest that the newly identified autosomal-recessive RHOH variant is associated with T cell dysfunction and recurrent opportunistic infections, functioning as a hypomorph by disrupting ZAP70-mediated TCR signaling. RHOH T cell TCR signaling opportunistic infection primary immunodeficiency Figures Figure 1 Figure 2 Figure 3 Introduction Ras homolog gene family H (RHOH) is an atypical member of the small GTPase family that lacks GTPase activity ( 1 ). It is specifically expressed in the hematopoietic system, with variable expression observed among different leukocyte subsets ( 2 ). Physiologically, RHOH is expressed in T, B, NK cells, and eosinophils in peripheral blood, but not in monocytes and neutrophils ( 2 , 3 ). RHOH is involved in T-cell receptor signaling through the recruitment and activation of ZAP70 and LCK ( 4 , 5 ). Disruption of RHOH (MIM: 602037) gene has been associated with various types of B-cell malignancies ( 4 ) and inborn errors of immunity (IEI) ( 6 ). Previously, a homozygous nonsense Y38X mutation in the RHOH gene (GenBank: NM_004310.5: c.114C > G, p.Tyr38Ter) was reported in siblings suffering from epidermodysplasia verruciformis, increased susceptibility to certain human papillomaviruses (HPV), lung disease, Burkitt lymphoma and impaired T-cell function (MIM: 618307) ( 6 ). Here we present a unique case of a 21-year-old male patient with recurrent infections affecting multiple organs, yet without any skin lesions. Genetic analysis identified a novel germline homozygous missense mutation in RHOH (p.Cys82Tyr), which, to the best of our knowledge, represents the first reported case of a missense variant in RHOH . Material and Methods Ethics statement Written informed consent was obtained from the patient and his parents. The study received approval from the institutional ethics review board of Huashan Hospital, Fudan University, and was conducted in accordance with the principles outlined in the Declaration of Helsinki. Whole exome sequencing and sanger sequencing Whole-exome sequencing was carried out using genomic DNA extracted from the whole blood of the patient and his parents using an Illunima HiSeq X Ten platform (2GB, 2×150bps paired-ends, 400×sequencing depth). Bioinformatics analysis was performed to detect rare sequence variants using the following databases: 1000 Genomes Project database, dbSNP, Exome Aggregation Consortium, and Genome Aggregation database. Functional prediction of variants was analyzed using the following bioinformatic tools: CADD, REVEL, SIFT, PolyPhen-2, and MutationTaster. Sanger sequencing was conducted to confirm the variant of the RHOH gene using the following primers: RHOH -F: 5’- TCTGCAAATCGCCGTCAGAG-3’, RHOH -R: 5’- CTGCTGTACTCCCCGATTGC-3’. HPV serology test Detection of HPV subtype 16 and 18 was performed using plasma from the patient and his parents by ELISA (Jianglai biological), according to the manufacturer’s instruction. All samples were assayed in triplicates. Antibodies The CD69 APC/Cy7 antibody (Biolegend) were used for flow cytometry. For immunoblot and immunoprecipitation analysis, the following antibodies were employed: polyclonal anti-RHOH (Abcam, ab118507), monoclonal anti-HA-tag (CST, clone 6E2), anti-ZAP70 (CST, clone L1E5) and anti-β-actin (CST, clone 13E5). For detection of the primary antibodies, the following HRP-conjugated secondary antibodies were used: goat anti-rabbit IgG (Proteintech, SA00001), conformational specific mouse anti-rabbit IgG (CST, 5127), and rat anti-mouse IgG (CST, 7076). In functional studies, the following antibodies were utilized: anti-CD3 antibody (Invitrogen, clone OKT3) and anti-CD28 antibody (Invitrogen, clone CD28.2). Plasmid construction and ectopic expression of RHOH For the transduction of Jurkat cells, full-length wild-type (WT) RHOH cDNA and the C82Y variant, with or without HA-tag, were generated by overlap extension PCR using PrimeSTAR Max DNA Polymerase (Takara) and inserted into the pMX-IRES-GFP vector. Immunoprecipitation and immunoblot analysis The constructed plasmids described above were transfected into HEK293 cells using Hieff Trans Liposomal Transfection Reagent (Yeason). Jurkat cells were transduced with retrovirus-expressing HA-tagged RHOH WT or RHOH C82Y , and sorted for GFP + cells. For immunoblot analysis of RHOH, GFP + Jurkat cells were lysed on ice with NP-40 buffer (Beyotime) supplemented with a protease and phosphatase inhibitor cocktail (Beyotime). The lysates were separated using a FuturePAGE 4–20% SDS-PAGE gel (ACE Biotechnology), and transferred to 0.22µm PVDF membranes (Beyotime). The membranes were blocked using non-fat milk, probed with primary antibodies overnight at 4℃, rinsed, and then incubated with HRP-conjugated secondary antibodies. The blots were developed using the Immobilon Western HRP substrate kit (Merck). For immunoprecipitation analysis, cells were lysed on ice with Mg2 + lysis wash buffer (in-house, containing 750 mM NaCl, 5% lgepal CA-630, 50 mM MgCl 2 , 5 mM EDTA, 10% glycerol and 125 mM HEPES, pH 7.5.) containing phosphatase inhibitors and protease cocktail. Per 500 µL of lysate supernatant was incubated with 50 µL anti-HA-tag antibody at 4℃ overnight and mixed with Protein A magnetic beads (Thermo Fisher). The resulting immunoprecipitants were separated by 4–20% SDS-PAGE gel, and analyzed by immunoblot. Quantitative PCR Total RNA was extracted from peripheral blood and cell lines using TRIzol reagent (Invitrogen). cDNA was synthesized using PrimeScript RT Master Mix (Takara). Quantitative real-time PCR was performed using TB Green Premix Ex Taq II kit (Takara). The relative expression of RHOH was calculated using GAPDH as an internal control. Primers for RHOH and GAPDH were as follow: RHOH -qPCR-F 5’-CAATGACGCCTTCAGAAGCA-3’, RHOH -qPCR-R 5’-ACAGGGGTACAGGGCAAGTT-3’; GAPDH-F 5’-CTGGGCTACACTGAGCACC-3’, GAPDH-R 5’-AAGTGGTCGTTGAGGGCAATG-3’. T-cell stimulation PBMCs were isolated from the proband and his parents and stored at -80°C until use. The cells were thawed and allowed to rest in RPMI 1640 supplemented with 10% fetal bovine serum for 30 min. The cells were then stimulated with 0.5 µg/mL anti-CD3 plus 2.5 µg/mL anti-CD28 antibodies, and analyzed using flow cytometry before and 4 days after stimulation. For analysis of CD69 up-regulation and RHOH interaction with ZAP70, Jurkat cells were stimulated pre-coated anti-CD3 antibody (10ug/ml). For analysis of IL-2, Jurkat cells were stimulated with plated-bound anti-CD3 (2 µg/mL) plus anti-CD28 (5 µg/mL) at 37℃ for 24 hours. CD69 up-regulation assay The up-regulation of CD69 was assessed in Jurkat cells expressing HA-tagged RHOH WT or RHOH C82Y , or an empty vector (EV) control. Jurkat cells were plated at 2.5×10 5 /well in a 24-well plate coated with anti-CD3 antibody. After 0 and 4 hours of stimulation, cells were collected and the expression of CD69 were analyzed by flow cytometry. ELISA The supernatants of stimulated Jurkat cells were collected. IL-2 cytokine production was detected by ELISA (LAIZEE) according to the manufacturer’s instruction. All samples were assayed in duplicates. Statistical analysis Data were analyzed using one-way ANOVA followed by Bonferroni's multiple comparisons test or two-tailed unpaired Student’s t tests with the GraphPad Prism 8 software (Dotmatics, USA). P < 0.05 was considered statistically significant. Results Clinical features of the patient Upon admission to our hospital, the patient had been suffering from recurrent lung infections and deterioration of eyesight for over a year. He had previously been in good health until his first episode of fever (peaking at 38 o C) accompanied by a productive cough in September 2018. His chest CT revealed infiltrations in both lungs. Laboratory tests revealed a normal white blood cell count (7.41×10 9 /L, reference range: 4–10×10 9 /L), an elevated C-reactive protein level (43.13 mg/L, reference range: <10 mg/L), and a severely reduced serum IgG level (0.8 g/L, reference range: 7.0–16.0 g/L). Immunological tests indicated a normal CD4 + T cell number, but a decreased CD4:CD8 ratio (Table 1 ). The proportions of B cells (0.9%, reference range: 6.48–16.64%) and NK cells (3.5%, reference range: 5.17–24.65%) were both below average. His HIV serology was negative. Culture and nucleic acid amplification tests of bacteria and fungus using the bronchoalveolar lavage fluid were all negative. After empirical antibiotic therapy and intravenous immunoglobulin (IVIg) infusion, he showed clinical improvement and was discharged. He was prescribed a one-week oral regimen of sulfamethoxazole and trimethoprim (SMZ-TMP) and cefaclor, along with monthly IVIg. Table 1 Immunological characteristics of the patient during 2018–2019 Immunologic parameter September 2018 Disease onset January 2019 CMV retinitis May 2019 Recurrent lung infection September 2019 Recurrent lung infection November 2019 Recurrent lung infection White blood cell (cells/µl) 8880 (3500–9500) 2260 7420 2800 ( 3500–9500) ** Lymphocyte (%) 42.7 (20–50) 31.48 (27.90–37.30) 34.5 (27.9–37.3) 29.8 35.4 (20.0–50.0) ** CD3 + T cells (cells/µl) 3575.6 1381 (1185–1901) 723 (1185–1901) 2023 [91.5%] § 787 [79.43%] # CD4 + T cells (cells/µl) 409.5 (200–1820) 270 (561–1137) 92 (561–1137) 464 [20.1%] § 206 [20.83%] # CD8 + T cells (cells/µl) 3025.8 (130–1350) 1067 (464–754) 522 (404–754) 1410 [63.8%] § 536 [54.03%] # CD4:CD8 ratio 0.14 (0.78–1.89) 0.25 (0.89–2.01) 0.18 (0.95–2.1) 0.33 0.39 (0.9–3.6) CD19 + B cells (cells/µl) 34.1 (50–670) 93 (180–324) 11 (180–324) 42 [1.9%] § 61 [6.20%] # CD56 + NK cells (cells/µl) 132.7 (40-1000) 107 (175–567) 46 (175–567) 130 [5.9%] § 244 [14.19%] # IgA (g/L) 0.92 (0.7-4.0) 0.5 (0.8-4.0) 0.38 (0.7-4.0) 0.29 (0.7-4.0) 0.34 (0.7-4.0) IgG (g/L) 0.8 (7.0–16.0) 5.0 * (7.0–15.0) 7.15 * (7.0–17.0) 0.87 (7.0–16.0) 3.15 * (7.0–16.0) IgM (g/L) 0.92 (0.4–2.3) 0.90 (0.4–2.4) 0.43 (0.4–2.3) 0.54 (0.4–2.3) 1.03 (0.4–2.3) CD4 + CD28 + /CD4 + (%) 58.8 (85.0-100.0) CD8 + CD28 + /CD8 + (%) 3.4 (37.2–60.4) CD8 + HLA-DR + /CD8 + (%) 82.5 (6.3–23.8) CD8 + CD38 + /CD8 + (%) 98.5 (32.4–57.4) Numbers in parentheses represent reference ranges. * Affected by IVIg supplementation. ** WBC number, proportion of lymphocyte and immune cell subset assay were determined using different samples. § Calculated based on subset proportions indicated in brackets. Reference ranges unavailable for subset proportions. # Cell numbers calculated based on subset proportions indicated in brackets. Reference ranges for subset proportions: CD3 + %: 49.1–83.6%, CD4 + %: 28.2–62.8%, CD8 + %: 10.2–40.1%, CD19 + %: 6–25%, Total NK%: 5–27%. From November 2018 to March 2019, the patient experienced a deterioration in visual acuity, xanthopsia and distorted vision, first in the right eye and subsequently in the left. A diagnoses of cytomegalovirus (CMV) retinitis was considered and intravitreal injections of ganciclovir were then initiated. However, the visual acuity of both eyes continued to deteriorate. He developed a headache during this period, and a brain MRI revealed neurocysticercosis of the right cerebellar hemisphere. His symptoms improved after the administration of praziquantel. Follow-up His follow-up head MR images are showed in Fig. 1 C. A hypointensity lesion (indicated with a white arrow) in the cingulate gyrus cortex of the right frontal lobe was demonstrated on both T1-weighted (T1W) and FLAIR images, and no T1W contrast enhancement were observed. Since May 2019, the patient experienced recurrent lung infections over the following 6 months. Chest CT images demonstrated diffuse exudation and reticulation in both lungs, particularly in lower lobes (Fig. 1 B). Next-generation sequencing of the bronchoalveolar lavage fluid detected Klebsiella aerogenes (reads 8), Pneumocystis jirovecii (reads 486) and rubella virus (RV) (reads 158). CMV DNA was detected in peripheral blood (1.29×10 4 copies/mL). His eyesight continued to deteriorate due to persistent CMV retinitis. Fundoscopy showed large patches of white granular retinal necrosis lesions and vascular occlusion in both eyes. Ocular ultrasonography showed vitreous opacities and retinal detachment in the right eye (Fig. 1 A). Next-generation sequencing of the aqueous humor identified CMV (reads 16), RV (reads 3) and Streptococcus equi (reads 2874). Throughout this period, a series of immunological tests revealed persistent CD4 + T cell lymphopenia and hypoimmunoglobulinemia (Table 1 ). The clinical possibility of acquired immunodeficiency was considered but later ruled out as tests for anti-IFN-γ autoantibody and HIV RNA were both negative. The lung infection resolved by a combined therapy of meropenem, ganciclovir, SMZ-TMP, caspofungin, and IVIg. Following discharge, the patient remained well for two years, but unfortunately succumbed to an unmanageable central nerve system infection in 2021. The patient is from a consanguineous family, with both parents being healthy. His sibling also suffered from recurrent severe lung infections. During her last episode, strong broad-spectrum antimicrobial agents, including cefoperazone/sulbactam, meropenem, linezolid, ganciclovir and voriconazole were used to treat the bacterial (pulmonary consolidation and infiltration lesions on chest CT), CMV (blood CMV DNA 2.06×10 2 copies) and suspected fungal (positive results of blood G test at different time points) infections in the lungs. Despite the implementation of IVIg and mechanical ventilation, she kept deteriorating, and eventually died of severe respiratory infection. Immunological features of the patient During the course of disease, he showed a persistent deficient in both humoral and cellular immunity. As shown in Table 1 , serial immunological tests conducted during 2018–2019 revealed that the patient exhibited persistently reduced numbers of CD4 + T and B cells, profound hypoimmunoglobulinemia, and sometimes a decreased number of NK cells. A similar pattern of T-cell lymphopenia (CD3 + CD4 + /CD45 + 14.8%, reference range: 30.0–54.0%) and hypoimmunoglobulinemia (IgG <0.333 g/L, reference range: 7.51–15.60 g/L; IgA 0.16 g/L, reference range: 0.82–4.53 g/L; IgM 0.10 g/L, reference range: 0.46–3.04 g/L) was also observed in his sister. The patient showed increased number of CD8 + T cells, which showed a more activated and differentiated phenotype as indicated by an elevated proportion of HLA-DR + or CD38 + cells( 7 ). Both the CD4 + and CD8 + T-cell subsets displayed a significantly lower proportion of CD28 + cells, suggesting a decrease in naïve T cell which express a high level of differentiation marker CD28( 7 ). Due to the limited availability of samples collected prior to the patient’s passing, further detailed immunological analysis of the patient could not be conducted. Identification of a novel germline missense mutation in RHOH The patient’s parents were consanguineous cousins, and both were in good health. However, the patient’s elder sister died at her 20s due to uncontrolled lung infection. Based on the patient’s clinical characteristics and family history, autosomal recessive immunologic disorders were suspected. Whole-exome sequencing of the kindred was therefore performed. We identified a total of 15 nonsynonymous single nucleotide variants ( PRF1, HELLS, LRP1, PAH, BLM, RLTPR, CD226, LIG1, ALK, ADA, RHOH, EGFR, MET, DDX58, PTCH1 ) and 2 frameshift deletions ( MICA, BTNL2 ) in the exons of protein-coding genes with a minor allele frequency lower than 0.01 in population databases, including 1000 Genomes Project database, dbSNP, Exome Aggregation Consortium or Genome Aggregation Database. Among these rare variants, the variant in RHOH is the only homozygous one in the patient. Both parents are heterozygous carriers of this variant, supporting an autosomal-recessive inheritance pattern for this novel germline RHOH mutation (Fig. 2 A). The variant (GenBank: NM_004310.5: c.245G > A, p.Cys82Tyr) resulted in substitution of an evolutionary conserved cysteine residue with a tyrosine residue in the ITAM-like domain responsible for interaction with ZAP70 (Fig. 2 B). The C82Y RHOH variant has not been reported in population databases. It is predicted to be pathogenic by multiple in silico pathogenicity prediction tools, including CADD (score = 26.4), REVEL (0.831, pathogenic), SIFT (0.001, deleterious), PolyPhen-2 (1.0, probably damaging), and MutationTaster (1, disease causing). Unfortunately, genetic assays of the patient’s sibling could not be performed due to the unavailability of a specimen. HPV infection was found in previous reported RHOH deficiency cases, but HPV serology test results of our patient and his parents were negative. Expression and function of RHOH C82Y We first assessed the effect of the C82Y variant on the mRNA expression of RHOH gene using quantitative-PCR (Fig. 3 A). The results showed that RHOH mRNA expression in peripheral blood was comparable to that of his parents and the controls. An impaired T cell activation has been reported in previous RHOH deficiency cases ( 6 ), we therefore analyzed how T cells respond to anti-CD3/CD28 stimulation in these kindred. As shown in Fig. 3 B, PBMC from the patient contained a much lower proportion of viable cells than did his parents, with or without TCR stimulation. The patient’s T cells failed to enlarge, as indicated by the significantly lower FSC of viable cells compared to his parents, suggesting a general defect in TCR-mediated T cell activation in the patient. Detailed immunologic analysis of the patient could not be done, because the patient succumbed to infection, and no more biological specimens were available for investigation. We therefore evaluate the expression and function of the C82Y RHOH variant using cell lines generated to express RHOH WT or RHOH C82Y . Comparable levels of wild type and mutant RHOH protein were observed in HEK293 cell line. However, the ectopic expression of HA-tagged RHOH in Jurkat cells revealed significantly compromised protein expression by the C82Y variant (Fig. 3 C). The C82Y variant maps to the ITAM domain of RHOH which interacts with ZAP70, a key molecule in TCR signaling pathway ( 5 ).To assess whether the C82Y variant disrupt the interaction between RHOH and ZAP70, co-immunoprecipitation was performed using Jurkat cells lysate (Fig. 3 D). In RHOH WT -expressing Jurkat cells, ZAP70 could bind with RHOH in a resting state, and RHOH-bound ZAP70 increased after anti-CD3 stimulation, consistent with previous publications( 5 ). Meanwhile, in Jurkat cells ectopically expressing RHOH C82Y , the protein expression level of the C82Y variant was significantly lower than that of the wild type, even after enrichment. More importantly, the binding between ZAP70 and RHOH was undetectable, both before and after anti-CD3 stimulation. Therefore, our findings demonstrate that the C82Y variant impairs RHOH function by undermining its interaction with downstream signaling molecule ZAP70. To verify whether the C82Y variant disrupted the signal transduction of TCR, we compared the expression of T-cell activation marker CD69 ( 8 ) in Jurkat cells ectopically expressing RHOH WT , the C82Y variant, or an empty vector control (EV) upon anti-CD3 stimulation (Fig. 3 E). In comparison to EV, a rapid elevation of CD69 level was observed in Jurkat cells expressing RHOH WT . In contrast, the expression of CD69 in RHOH C82Y -expressing Jurkat cells failed to up-regulate, and remained at a level comparable to that of EV Jurkat cells. These observations suggest that the C82Y is a loss-of-function variant but does not exert a dominant negative effect over the endogenous WT RHOH. Measurement of IL-2 production revealed similar results (Fig. 3 F). The culture supernatants of Jurkat cells expressing RHOH C82Y contained a much lower level of IL-2 than that of RHOH WT -expressing Jurkat cells. Collectively, these results indicate that the C82Y variant is a hypomorphic mutation which hampers RHOH protein expression and impairs its function in TCR-mediated T cell activation. Discussion The RHOH gene encodes a hematopoietic-specific Rho GTPase expressed in myeloid, NK, B, and T lymphocytes. As a membrane-bound adaptor for proximal protein kinases involved in TCR signal transduction, RHOH exerts a critical role in the activation, development and differentiation of T lymphocytes ( 1 , 4 ). Somatic mutations in RHOH gene have been linked to certain hematological malignancies ( 4 ). A germline mutation in RHOH has been identified as the cause of an autosomal recessive IEI ( 6 ). To date, only one kindred of RHOH deficiency, also called epidermodysplasia verruciformis-4, has been documented ( 6 ). The probands are two siblings from a consanguineous family. Both had a homozygous Y38X nonsense variant in RHOH gene. The Y38X variant led to a complete loss of RHOH protein expression. Both patients experienced early-onset disease. The proband, since the age of 5, developed Burkitt lymphoma, epidermodysplasia verruciformis, skin lesions due to HPV subtypes 3, 12, and 20, as well as pulmonary diseases, including a granulomatous lesion, chronic bronchial disease with emphysema, and right pneumothorax. His sister suffered from gingivostomatitis and similar treatment-resistant skin lesions caused by HPV subtype 20 from the age of 2. Our case presents a distinct clinical phenotype. Firstly, our patient experienced a late disease onset at the age of 21 years. Secondly, the typical skin lesion associated with epidermodysplasia verruciformis was absent, and no evidence of HPV infection was found. Thirdly, the patient exhibited recurrent infections affecting multiple organs, including the lungs, retina, and brain, caused by various opportunistic pathogens such as CMV, RV, P. jirovecii , helminth, and multiple bacteria. Similarly, his sibling also suffered from recurrent severe lung infections and free from HPV-related skin conditions. Our patient’s immunophenotype shared similarities with previously reported RHOH deficiency cases in terms of reduced CD4 + T cells, increased CD8 + T cells, and an elevated proportion of activated, differentiated effector and memory CD8 + T cells. Crequer, A. et al. revealed that the RHOH Y38X variant resulted in a complete absence of RHOH protein expression, defects in T-cell development and TCR signaling, impaired T-cell proliferation in response to certain antigens, and tissue-homing defects ( 6 ). In our case, the C82Y variant allows for normal transcription of the RHOH mutant, but disrupted its protein expression. The impaired response to TCR stimulation of the patient T cell was consistent with previous RHOH deficiency cases. The defective upregulation of CD69 and production of IL-2 in RHOH C82Y -expressing Jurkat cells also support that the C82Y variant is functionally impaired, particularly in TCR-induced T cell activation. Studies on RhoH −/− mice have observed a defect in thymocyte selection, maturation, lower T-cell cytotoxicity and cytokine release, and have demonstrated that it is a result of reduced activation of ZAP70-mediated signaling and impaired translocation of ZAP70 to the immunological synapse ( 5 , 9 ). The function of RHOH depends on the phosphorylation of its ITAMs ( 5 ). Considering that the C82Y variant is located within the ITAM-like motif, it is plausible that the defective function of the RHOH C82Y variant arises from its impaired interaction with ZAP70. Results from the immunoprecipitation analysis support our hypothesis, as RHOH-bound ZAP70 was undetectable in RHOH C82Y Jurkat cells. The C82Y variant led to a major defect in the interaction between RHOH and ZAP70, and impaired subsequent TCR signaling, at an extent similarly to what has been observed in cases with stop-gain RHOH mutation. Nevertheless, Dorn, T., et al. found that RHOH is not required for TCR-induced activation of ZAP70, but is indispensable for the efficient interaction of ZAP70 with the LAT signalosome ( 10 ). Since RHOH-bound ZAP70 was barely detected, we were unable to determine the phosphorylation and activation of ZAP70. Previous research also proposed that RHOH regulates T-cell development and function by maintaining LCK in an inactivated states ( 11 ), allowing T cells to switch between sensing chemokine-mediated go signals and TCR-dependent stop signals ( 12 ), and maintaining lymphocyte LFA-1 in a nonadhesive state ( 13 ). It also played a role in the differentiation of Th 17 cell by modulating the expression and function of RORγt ( 14 ). Our data provide an explanation for how the RHOH C82Y variant might be responsible for the T cell deficiency in this patient. But, further investigation is required, to understand the role of RHOH C82Y mutant in pre-TCR, TCR signaling and regulation of T-cell function. Our case also exhibited significant differences in the immunophenotype. In the previously reported cases, no obvious abnormalities were observed in B cells, NK cells, NKT cells, or antibody production. In contrast, our patient presented a persistent decrease in B and NK cell numbers, and profound hypoimmunoglobulinemia, indicating a combined defect in both cellular and humoral immune responses. This immunophenotype aligns with the expression pattern of RHOH in immune cells. The RHOH C82Y might also be associated with abnormality in BCR signaling, However, BCR signaling has also been found intact in Rhoh -deficient mice ( 15 ). A recent study in patients with CD28 deficiency strongly suggest that the poor antibody responses seen in those patients after vaccination results from a deficiency of CD4 T cell response, due to the absence of CD28 co-stimulation signaling ( 16 ). In order to provide an explanation for the pathogenesis of B and NK cell deficiency in our patient with the RHOH C82Y variant, further investigation using mouse model is required. In this study, we reported a novel homozygous germline variant in the RHOH gene (p.Cys82Tyr), associated with an autosomal recessive IEI. The affected individual exhibited recurrent, invasive, opportunistic infections in the lungs, eyes and brain during early adulthood, accompanied by a persistent decrease in CD4 + T, B, NK cells, and hypoimmunoglobulinemia. Immunological analysis demonstrated both reduced protein expression and defective binding of the C82Y mutant to the signaling molecule ZAP70. Impaired TCR-mediated T-cell activation was observed in both the patient and in RHOH C82Y -expressing Jurkat cells, indicating that the C82Y variant is a hypomorph associated with abnormalities in TCR signaling. Moreover, we confirmed that the C82Y variant does not exert a dominant negative effect. In conclusion, our case expands the genetic and phenotypic spectrum of RHOH deficiency and provides new insights into the development and regulation of T, B and NK cells. Declarations Funding This work was supported by the National Natural Science Foundation of China (grant #82271794 to Q-L R and grants #32330033 and #32270932 to J-YW). Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions J.Y. Zhou, M.Q. Qian participated in research investigation, data analysis and writing-original draft; J. Ning and J. Wu participated in research investigation, data analysis, contributed to methodology and software, writing-review & editing; X.Q. Feng, M.P. Yu and M. Qin participated in research investigation, contributed to methodology; H.X. Xu, Y.X. Yang, Q.L. Yang, F.R. Zhou, L.Y. Shao, H.X. Zhu and Y. Yang participated in clinical management and data collection of the kindred; Q.L. Ruan and J.Y. Wang: participated in research design, writing-editing and research funding, contributed research materials; W.H. Zhang participated in project administration. Data availability The exome sequencing data are not publicly available due to privacy restrictions. Ethics approval The study received approval from the institutional ethics review board of Huashan Hospital, Fudan University, and was conducted in accordance with the principles outlined in the Declaration of Helsinki. Consent to participate Informed consent was obtained from all individual participants included in the study. References Gu Y, Jasti AC, Jansen M, Siefring JE. RhoH, a hematopoietic-specific Rho GTPase, regulates proliferation, survival, migration, and engraftment of hematopoietic progenitor cells. Blood. 2005;105(4):1467-75. Schmidt-Mende J, Geering B, Yousefi S, Simon HU. Lysosomal degradation of RhoH protein upon antigen receptor activation in T but not B cells. Eur J Immunol. 2010;40(2):525-9. Stoeckle C, Geering B, Yousefi S, Rožman S, Andina N, Benarafa C, et al. RhoH is a negative regulator of eosinophilopoiesis. Cell Death Differ. 2016;23(12):1961-72. Ahmad Mokhtar AM, Hashim IF, Mohd Zaini Makhtar M, Salikin NH, Amin-Nordin S. The Role of RhoH in TCR Signalling and Its Involvement in Diseases. Cells. 2021;10(4). Gu Y, Chae HD, Siefring JE, Jasti AC, Hildeman DA, Williams DA. RhoH GTPase recruits and activates Zap70 required for T cell receptor signaling and thymocyte development. Nat Immunol. 2006;7(11):1182-90. Crequer A, Troeger A, Patin E, Ma CS, Picard C, Pedergnana V, et al. Human RHOH deficiency causes T cell defects and susceptibility to EV-HPV infections. J Clin Invest. 2012;122(9):3239-47. Mahnke YD, Brodie TM, Sallusto F, Roederer M, Lugli E. The who's who of T-cell differentiation: human memory T-cell subsets. Eur J Immunol. 2013;43(11):2797-809. Ziegler SF, Ramsdell F, Alderson MR. The activation antigen CD69. Stem Cells. 1994;12(5):456-65. Chae HD, Siefring JE, Hildeman DA, Gu Y, Williams DA. RhoH regulates subcellular localization of ZAP-70 and Lck in T cell receptor signaling. PLoS One. 2010;5(11):e13970. Dorn T, Kuhn U, Bungartz G, Stiller S, Bauer M, Ellwart J, et al. RhoH is important for positive thymocyte selection and T-cell receptor signaling. Blood. 2007;109(6):2346-55. Wang H, Zeng X, Fan Z, Lim B. RhoH modulates pre-TCR and TCR signalling by regulating LCK. Cell Signal. 2011;23(1):249-58. Baker CM, Comrie WA, Hyun YM, Chung HL, Fedorchuk CA, Lim K, et al. Opposing roles for RhoH GTPase during T-cell migration and activation. Proc Natl Acad Sci U S A. 2012;109(26):10474-9. Cherry LK, Li X, Schwab P, Lim B, Klickstein LB. RhoH is required to maintain the integrin LFA-1 in a nonadhesive state on lymphocytes. Nat Immunol. 2004;5(9):961-7. Tamehiro N, Nishida K, Sugita Y, Hayakawa K, Oda H, Nitta T, et al. Ras homolog gene family H (RhoH) deficiency induces psoriasis-like chronic dermatitis by promoting T(H)17 cell polarization. J Allergy Clin Immunol. 2019;143(5):1878-91. Sanchez-Aguilera A, Rattmann I, Drew DZ, Müller LU, Summey V, Lucas DM, et al. Involvement of RhoH GTPase in the development of B-cell chronic lymphocytic leukemia. Leukemia. 2010;24(1):97-104. Béziat V, Rapaport F, Hu J, Titeux M, Bonnet des Claustres M, Bourgey M, et al. Humans with inherited T cell CD28 deficiency are susceptible to skin papillomaviruses but are otherwise healthy. Cell. 2021;184(14):3812-28.e30. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 22 May, 2024 Read the published version in Journal of Clinical Immunology → Version 1 posted Editorial decision: Revision requested 28 Mar, 2024 Reviews received at journal 04 Mar, 2024 Reviewers agreed at journal 25 Feb, 2024 Reviewers invited by journal 22 Feb, 2024 Editor assigned by journal 19 Feb, 2024 Submission checks completed at journal 19 Feb, 2024 First submitted to journal 15 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3958385","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":273749785,"identity":"5ac3b25b-61f6-4570-8e83-61f2e7f17ebe","order_by":0,"name":"Jingyu Zhou","email":"","orcid":"","institution":"National Medical Center for Infectious Diseases, Huashan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Jingyu","middleName":"","lastName":"Zhou","suffix":""},{"id":273749786,"identity":"5cdd5c25-d6e7-4f31-b0bc-83f230e50812","order_by":1,"name":"Mengqing Qian","email":"","orcid":"","institution":"National Medical Center for Infectious Diseases, Huashan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Mengqing","middleName":"","lastName":"Qian","suffix":""},{"id":273749789,"identity":"f04565a2-647b-45b6-8794-6b1067e286b6","order_by":2,"name":"Ning Jiang","email":"","orcid":"","institution":"National Medical Center for Infectious Diseases, Huashan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Ning","middleName":"","lastName":"Jiang","suffix":""},{"id":273749790,"identity":"e3ae6700-e473-44ca-8c04-6888593bb55b","order_by":3,"name":"Jing Wu","email":"","orcid":"","institution":"National Medical Center for Infectious Diseases, Huashan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Wu","suffix":""},{"id":273749792,"identity":"4b3cc638-8aaf-492a-8715-68325858d35e","order_by":4,"name":"Xiaoqian Feng","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoqian","middleName":"","lastName":"Feng","suffix":""},{"id":273749794,"identity":"5261f8fe-69b5-4633-b8bd-e5162aadec9a","order_by":5,"name":"Meiping Yu","email":"","orcid":"","institution":"Children’s Hospital of Fudan University, National Children’s Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Meiping","middleName":"","lastName":"Yu","suffix":""},{"id":273749795,"identity":"ca2f51c5-c1ab-4add-bad6-b5c16229e3ee","order_by":6,"name":"Qing Min","email":"","orcid":"","institution":"Children’s Hospital of Fudan University, National Children’s Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Min","suffix":""},{"id":273749796,"identity":"bf88a8ba-0abb-43da-b59a-2cac68114fb7","order_by":7,"name":"Haoxin Xu","email":"","orcid":"","institution":"National Medical Center for Infectious Diseases, Huashan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Haoxin","middleName":"","lastName":"Xu","suffix":""},{"id":273749797,"identity":"5797cf62-6e8a-4f94-adb6-f5f93de1c68d","order_by":8,"name":"Yixuan Yang","email":"","orcid":"","institution":"National Medical Center for Infectious Diseases, Huashan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Yixuan","middleName":"","lastName":"Yang","suffix":""},{"id":273749798,"identity":"553aaff1-7ba4-40a7-a7bc-d3b202fde663","order_by":9,"name":"Qingluan Yang","email":"","orcid":"","institution":"National Medical Center for Infectious Diseases, Huashan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Qingluan","middleName":"","lastName":"Yang","suffix":""},{"id":273749799,"identity":"54ba5e08-23b6-4916-8ffa-bc29d6882514","order_by":10,"name":"Feiran Zhou","email":"","orcid":"","institution":"National Medical Center for Infectious Diseases, Huashan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Feiran","middleName":"","lastName":"Zhou","suffix":""},{"id":273749800,"identity":"2c8a23fa-e857-4aff-ab81-e69be7a93a19","order_by":11,"name":"Lingyun Shao","email":"","orcid":"","institution":"National Medical Center for Infectious Diseases, Huashan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Lingyun","middleName":"","lastName":"Shao","suffix":""},{"id":273749801,"identity":"d53b47f3-2004-4892-a7fe-cb797e8e9a8c","order_by":12,"name":"Haoxiang Zhu","email":"","orcid":"","institution":"National Medical Center for Infectious Diseases, Huashan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Haoxiang","middleName":"","lastName":"Zhu","suffix":""},{"id":273749802,"identity":"77dd47af-c774-43ee-a048-c3ab9eeda56b","order_by":13,"name":"Yun Yang","email":"","orcid":"","institution":"the First People’s Hospital of Yunnan Province","correspondingAuthor":false,"prefix":"","firstName":"Yun","middleName":"","lastName":"Yang","suffix":""},{"id":273749803,"identity":"e9a45523-be4a-4cb0-b958-c0784e739c51","order_by":14,"name":"Ji-Yang Wang","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Ji-Yang","middleName":"","lastName":"Wang","suffix":""},{"id":273749804,"identity":"d7e8f81e-4f6f-44c9-b6ee-40167c206c51","order_by":15,"name":"Qiaoling Ruan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIie2RMQrCMBhGEwLpktY1oahXqBQCYg8TEToLHkBB+CfFVW+hm6MSaJceoE7i4ubUyUEwdnRI6+aQN3y85U0fQg7HP+LVy+cbckK89lNTQupN8A7Ub0mK91nbJND+nT+PmsQZG5TPI+oFpcLV1JII7UmxLjSVGYuH6wLFolQk3FqSSFPKfdBMXlfSCBrvS0UJa0jECzSPgUkjaN4qCX1Io4gyaQSpqCkRmpJRFxLFMzozwge74rYMbUmQA748gKsOkIORpB/kk3NlS75f+FyDF7agxdcOh8PheANaREXDPQuZLQAAAABJRU5ErkJggg==","orcid":"","institution":"National Medical Center for Infectious Diseases, Huashan Hospital, Fudan University","correspondingAuthor":true,"prefix":"","firstName":"Qiaoling","middleName":"","lastName":"Ruan","suffix":""},{"id":273749806,"identity":"d8e51a21-518f-4581-9f85-adc3ba77512b","order_by":16,"name":"Wenhong Zhang","email":"","orcid":"","institution":"National Medical Center for Infectious Diseases, Huashan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Wenhong","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-02-15 10:44:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3958385/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3958385/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10875-024-01735-4","type":"published","date":"2024-05-22T08:15:52+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51443988,"identity":"a88e665e-0e1c-457e-a9ff-af1f84fec369","added_by":"auto","created_at":"2024-02-21 18:02:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3288689,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImages of the proband in 2019. (A) \u003c/strong\u003eFundoscopy showed large patches of white granular retinal necrosis lesions and vascular occlusion in both eyes (Left and middle panels). Ocular ultrasonography showed vitreous opacities and retinal detachment in the right eye (Right panel). \u003cstrong\u003e(B)\u003c/strong\u003e Chest CT images showed diffuse exudation and reticulation in both lungs, particularly in lower lobes. \u003cstrong\u003e(C)\u003c/strong\u003e Head MR images demonstrated a hypointensity lesion (indicated with a white arrow) on both T1-weighted (T1W) and FLAIR images, and no T1W contrast enhancement were observed.\u003c/p\u003e","description":"","filename":"FIG1.png","url":"https://assets-eu.researchsquare.com/files/rs-3958385/v1/9f49afc02ea82020770d645b.png"},{"id":51443990,"identity":"af411aec-9b00-437f-bc02-c945484f8b33","added_by":"auto","created_at":"2024-02-21 18:02:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":401296,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of a novel germline missense mutation in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eRHOH\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. (A)\u003c/strong\u003eFamily Pedigree of the patient, and Sanger sequencing results of the novel \u003cem\u003eRHOH\u003c/em\u003e mutation (c.245G\u0026gt;A, p.Cys82Tyr) in the family. The proband is indicated by a filled pattern with a black arrow. Genotype of the sibling is unknown due to inaccessibility of sample. \u003cstrong\u003e(B)\u003c/strong\u003e Top: Schematic diagram of RHOH domain architecture (UniProt accession number: Q15669.1). The C82Y variant is indicated by a red arrow. The previously reported nonsense mutation (c.114C\u0026gt;G, p.Tyr38Ter) identified in autosomal-recessive epidermodysplasia verruciformis is indicated by a black arrow. Bottom: Alignment of the amino acids in the ITAM-like motif of RHOH from 5 different species. The highly conservative C82 is indicated in a red box.\u003c/p\u003e","description":"","filename":"FIG2.png","url":"https://assets-eu.researchsquare.com/files/rs-3958385/v1/02803cdb28f099a14eeba448.png"},{"id":51443991,"identity":"032559df-40d2-4817-958e-4237febd1bd3","added_by":"auto","created_at":"2024-02-21 18:02:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1466013,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression and function of RHOH\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eC82Y\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e. (A) \u003c/strong\u003eThe relative expression of \u003cem\u003eRHOH\u003c/em\u003e in the patient, his parents and controls; \u003cem\u003eGAPDH\u003c/em\u003e served as an internal control. \u003cstrong\u003e(B)\u003c/strong\u003e PBMCs from the patient and his parents were thawed and stimulated with anti-CD3 plus anti-CD28 antibodies. The activation of T cell was assessed by the proportion and FSC of viable cells before and 4 days after stimulation.\u003cstrong\u003e (C) \u003c/strong\u003eRHOH protein expression in HEK293\u003cstrong\u003e \u003c/strong\u003ecells transfected with wild-type (WT) or C82Y \u003cem\u003eRHOH\u003c/em\u003e plasmids (left), and Jurkat cells transduced with retrovirus expressing HA-tagged RHOH\u003csup\u003eWT\u003c/sup\u003e or RHOH\u003csup\u003eC82Y\u003c/sup\u003e, empty vector (EV) control, or untreated (UT) Jurkat cells (right). β-actin served as a loading control.\u003cstrong\u003e (D)\u003c/strong\u003e Jurkat cells expressing RHOH\u003csup\u003eWT\u003c/sup\u003e or RHOH\u003csup\u003eC82Y\u003c/sup\u003e were stimulated with pre-coated anti-CD3 antibody, and cell lysates were subject to immunoprecipitation analysis. Binding affinity was determined by the ratio of bound total ZAP70 to HA-tagged RHOH.\u003cstrong\u003e (E) \u003c/strong\u003eExpression of CD69 in\u003cstrong\u003e \u003c/strong\u003eJurkat cells expressing EV control, RHOH\u003csup\u003eWT\u003c/sup\u003e or RHOH\u003csup\u003eC82Y\u003c/sup\u003e, or untreated Jurkat cells after 4-hour anti-CD3 stimulation. \u003cstrong\u003e(F)\u003c/strong\u003e IL-2 production in cultural supernatants of Jurkat cells expressing EV control, RHOH\u003csup\u003eWT\u003c/sup\u003e or RHOH\u003csup\u003eC82Y\u003c/sup\u003e, or untreated Jurkat cells after anti-CD3/CD28 stimulation. Data are presented as mean ± SD or individual values. ns, not significant; *, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"FIG3.png","url":"https://assets-eu.researchsquare.com/files/rs-3958385/v1/2f083c157d1e1ffc6a3e8878.png"},{"id":59253733,"identity":"9101011d-d3fa-419f-a6a8-78ba9467888b","added_by":"auto","created_at":"2024-06-28 08:16:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8843706,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3958385/v1/111fd651-463a-48e9-8dda-59e4fb7d7724.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A novel homozygous RHOH variant associated with T cell dysfunction and recurrent opportunistic infections","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRas homolog gene family H (RHOH) is an atypical member of the small GTPase family that lacks GTPase activity (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). It is specifically expressed in the hematopoietic system, with variable expression observed among different leukocyte subsets (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Physiologically, \u003cem\u003eRHOH\u003c/em\u003e is expressed in T, B, NK cells, and eosinophils in peripheral blood, but not in monocytes and neutrophils (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). RHOH is involved in T-cell receptor signaling through the recruitment and activation of ZAP70 and LCK (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Disruption of \u003cem\u003eRHOH\u003c/em\u003e (MIM: 602037) gene has been associated with various types of B-cell malignancies (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) and inborn errors of immunity (IEI) (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Previously, a homozygous nonsense Y38X mutation in the \u003cem\u003eRHOH\u003c/em\u003e gene (GenBank: NM_004310.5: c.114C\u0026thinsp;\u0026gt;\u0026thinsp;G, p.Tyr38Ter) was reported in siblings suffering from epidermodysplasia verruciformis, increased susceptibility to certain human papillomaviruses (HPV), lung disease, Burkitt lymphoma and impaired T-cell function (MIM: 618307) (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Here we present a unique case of a 21-year-old male patient with recurrent infections affecting multiple organs, yet without any skin lesions. Genetic analysis identified a novel germline homozygous missense mutation in \u003cem\u003eRHOH\u003c/em\u003e (p.Cys82Tyr), which, to the best of our knowledge, represents the first reported case of a missense variant in \u003cem\u003eRHOH\u003c/em\u003e.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003eWritten informed consent was obtained from the patient and his parents. The study received approval from the institutional ethics review board of Huashan Hospital, Fudan University, and was conducted in accordance with the principles outlined in the Declaration of Helsinki.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eWhole exome sequencing and sanger sequencing\u003c/h2\u003e \u003cp\u003eWhole-exome sequencing was carried out using genomic DNA extracted from the whole blood of the patient and his parents using an Illunima HiSeq X Ten platform (2GB, 2\u0026times;150bps paired-ends, 400\u0026times;sequencing depth). Bioinformatics analysis was performed to detect rare sequence variants using the following databases: 1000 Genomes Project database, dbSNP, Exome Aggregation Consortium, and Genome Aggregation database. Functional prediction of variants was analyzed using the following bioinformatic tools: CADD, REVEL, SIFT, PolyPhen-2, and MutationTaster.\u003c/p\u003e \u003cp\u003eSanger sequencing was conducted to confirm the variant of the \u003cem\u003eRHOH\u003c/em\u003e gene using the following primers: \u003cem\u003eRHOH\u003c/em\u003e-F: 5\u0026rsquo;- TCTGCAAATCGCCGTCAGAG-3\u0026rsquo;, \u003cem\u003eRHOH\u003c/em\u003e-R: 5\u0026rsquo;- CTGCTGTACTCCCCGATTGC-3\u0026rsquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHPV serology test\u003c/h2\u003e \u003cp\u003eDetection of HPV subtype 16 and 18 was performed using plasma from the patient and his parents by ELISA (Jianglai biological), according to the manufacturer\u0026rsquo;s instruction. All samples were assayed in triplicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies\u003c/h2\u003e \u003cp\u003eThe CD69 APC/Cy7 antibody (Biolegend) were used for flow cytometry. For immunoblot and immunoprecipitation analysis, the following antibodies were employed: polyclonal anti-RHOH (Abcam, ab118507), monoclonal anti-HA-tag (CST, clone 6E2), anti-ZAP70 (CST, clone L1E5) and anti-β-actin (CST, clone 13E5). For detection of the primary antibodies, the following HRP-conjugated secondary antibodies were used: goat anti-rabbit IgG (Proteintech, SA00001), conformational specific mouse anti-rabbit IgG (CST, 5127), and rat anti-mouse IgG (CST, 7076). In functional studies, the following antibodies were utilized: anti-CD3 antibody (Invitrogen, clone OKT3) and anti-CD28 antibody (Invitrogen, clone CD28.2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid construction and ectopic expression of RHOH\u003c/h2\u003e \u003cp\u003eFor the transduction of Jurkat cells, full-length wild-type (WT) \u003cem\u003eRHOH\u003c/em\u003e cDNA and the C82Y variant, with or without HA-tag, were generated by overlap extension PCR using PrimeSTAR Max DNA Polymerase (Takara) and inserted into the pMX-IRES-GFP vector.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunoprecipitation and immunoblot analysis\u003c/h2\u003e \u003cp\u003eThe constructed plasmids described above were transfected into HEK293 cells using Hieff Trans Liposomal Transfection Reagent (Yeason). Jurkat cells were transduced with retrovirus-expressing HA-tagged \u003cem\u003eRHOH\u003c/em\u003e\u003csup\u003eWT\u003c/sup\u003e or \u003cem\u003eRHOH\u003c/em\u003e\u003csup\u003eC82Y\u003c/sup\u003e, and sorted for GFP\u003csup\u003e+\u003c/sup\u003e cells. For immunoblot analysis of RHOH, GFP\u003csup\u003e+\u003c/sup\u003e Jurkat cells were lysed on ice with NP-40 buffer (Beyotime) supplemented with a protease and phosphatase inhibitor cocktail (Beyotime). The lysates were separated using a FuturePAGE 4\u0026ndash;20% SDS-PAGE gel (ACE Biotechnology), and transferred to 0.22\u0026micro;m PVDF membranes (Beyotime). The membranes were blocked using non-fat milk, probed with primary antibodies overnight at 4℃, rinsed, and then incubated with HRP-conjugated secondary antibodies. The blots were developed using the Immobilon Western HRP substrate kit (Merck).\u003c/p\u003e \u003cp\u003eFor immunoprecipitation analysis, cells were lysed on ice with Mg2\u003csup\u003e+\u003c/sup\u003e lysis wash buffer (in-house, containing 750 mM NaCl, 5% lgepal CA-630, 50 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 5 mM EDTA, 10% glycerol and 125 mM HEPES, pH 7.5.) containing phosphatase inhibitors and protease cocktail. Per 500 \u0026micro;L of lysate supernatant was incubated with 50 \u0026micro;L anti-HA-tag antibody at 4℃ overnight and mixed with Protein A magnetic beads (Thermo Fisher). The resulting immunoprecipitants were separated by 4\u0026ndash;20% SDS-PAGE gel, and analyzed by immunoblot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from peripheral blood and cell lines using TRIzol reagent (Invitrogen). cDNA was synthesized using PrimeScript RT Master Mix (Takara). Quantitative real-time PCR was performed using TB Green Premix Ex Taq II kit (Takara). The relative expression of \u003cem\u003eRHOH\u003c/em\u003e was calculated using \u003cem\u003eGAPDH\u003c/em\u003e as an internal control. Primers for \u003cem\u003eRHOH\u003c/em\u003e and \u003cem\u003eGAPDH\u003c/em\u003e were as follow: \u003cem\u003eRHOH\u003c/em\u003e-qPCR-F 5\u0026rsquo;-CAATGACGCCTTCAGAAGCA-3\u0026rsquo;, \u003cem\u003eRHOH\u003c/em\u003e-qPCR-R 5\u0026rsquo;-ACAGGGGTACAGGGCAAGTT-3\u0026rsquo;; GAPDH-F 5\u0026rsquo;-CTGGGCTACACTGAGCACC-3\u0026rsquo;, GAPDH-R 5\u0026rsquo;-AAGTGGTCGTTGAGGGCAATG-3\u0026rsquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eT-cell stimulation\u003c/h2\u003e \u003cp\u003ePBMCs were isolated from the proband and his parents and stored at -80\u0026deg;C until use. The cells were thawed and allowed to rest in RPMI 1640 supplemented with 10% fetal bovine serum for 30 min. The cells were then stimulated with 0.5 \u0026micro;g/mL anti-CD3 plus 2.5 \u0026micro;g/mL anti-CD28 antibodies, and analyzed using flow cytometry before and 4 days after stimulation. For analysis of CD69 up-regulation and RHOH interaction with ZAP70, Jurkat cells were stimulated pre-coated anti-CD3 antibody (10ug/ml). For analysis of IL-2, Jurkat cells were stimulated with plated-bound anti-CD3 (2 \u0026micro;g/mL) plus anti-CD28 (5 \u0026micro;g/mL) at 37℃ for 24 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCD69 up-regulation assay\u003c/h2\u003e \u003cp\u003eThe up-regulation of CD69 was assessed in Jurkat cells expressing HA-tagged RHOH\u003csup\u003eWT\u003c/sup\u003e or RHOH\u003csup\u003eC82Y\u003c/sup\u003e, or an empty vector (EV) control. Jurkat cells were plated at 2.5\u0026times;10\u003csup\u003e5\u003c/sup\u003e/well in a 24-well plate coated with anti-CD3 antibody. After 0 and 4 hours of stimulation, cells were collected and the expression of CD69 were analyzed by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eThe supernatants of stimulated Jurkat cells were collected. IL-2 cytokine production was detected by ELISA (LAIZEE) according to the manufacturer\u0026rsquo;s instruction. All samples were assayed in duplicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using one-way ANOVA followed by Bonferroni's multiple comparisons test or two-tailed unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e tests with the GraphPad Prism 8 software (Dotmatics, USA). \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eClinical features of the patient\u003c/h2\u003e \u003cp\u003eUpon admission to our hospital, the patient had been suffering from recurrent lung infections and deterioration of eyesight for over a year. He had previously been in good health until his first episode of fever (peaking at 38\u003csup\u003eo\u003c/sup\u003eC) accompanied by a productive cough in September 2018. His chest CT revealed infiltrations in both lungs. Laboratory tests revealed a normal white blood cell count (7.41\u0026times;10\u003csup\u003e9\u003c/sup\u003e /L, reference range: 4\u0026ndash;10\u0026times;10\u003csup\u003e9\u003c/sup\u003e /L), an elevated C-reactive protein level (43.13 mg/L, reference range: \u0026lt;10 mg/L), and a severely reduced serum IgG level (0.8 g/L, reference range: 7.0\u0026ndash;16.0 g/L). Immunological tests indicated a normal CD4\u003csup\u003e+\u003c/sup\u003e T cell number, but a decreased CD4:CD8 ratio (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The proportions of B cells (0.9%, reference range: 6.48\u0026ndash;16.64%) and NK cells (3.5%, reference range: 5.17\u0026ndash;24.65%) were both below average. His HIV serology was negative. Culture and nucleic acid amplification tests of bacteria and fungus using the bronchoalveolar lavage fluid were all negative. After empirical antibiotic therapy and intravenous immunoglobulin (IVIg) infusion, he showed clinical improvement and was discharged. He was prescribed a one-week oral regimen of sulfamethoxazole and trimethoprim (SMZ-TMP) and cefaclor, along with monthly IVIg.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eImmunological characteristics of the patient during 2018\u0026ndash;2019\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eImmunologic parameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeptember 2018 \u003c/p\u003e \u003cp\u003eDisease onset\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJanuary 2019\u003c/p\u003e \u003cp\u003eCMV retinitis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMay 2019\u003c/p\u003e \u003cp\u003eRecurrent lung infection\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSeptember 2019\u003c/p\u003e \u003cp\u003eRecurrent lung infection\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNovember 2019\u003c/p\u003e \u003cp\u003eRecurrent lung infection\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWhite blood cell (cells/\u0026micro;l)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8880 (3500\u0026ndash;9500)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2260\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7420\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2800 ( 3500\u0026ndash;9500)\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymphocyte (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.7 (20\u0026ndash;50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.48 (27.90\u0026ndash;37.30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.5 (27.9\u0026ndash;37.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e29.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e35.4 (20.0\u0026ndash;50.0)\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD3\u003csup\u003e+\u003c/sup\u003e T cells (cells/\u0026micro;l)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3575.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1381 (1185\u0026ndash;1901)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e723 (1185\u0026ndash;1901)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2023 [91.5%]\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e787 [79.43%]\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells (cells/\u0026micro;l)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e409.5 (200\u0026ndash;1820)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e270 (561\u0026ndash;1137)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92 (561\u0026ndash;1137)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e464 [20.1%]\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e206 [20.83%]\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells (cells/\u0026micro;l)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3025.8 (130\u0026ndash;1350)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1067 (464\u0026ndash;754)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e522 (404\u0026ndash;754)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1410 [63.8%]\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e536 [54.03%]\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD4:CD8 ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.14 (0.78\u0026ndash;1.89)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.25 (0.89\u0026ndash;2.01)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.18 (0.95\u0026ndash;2.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.39 (0.9\u0026ndash;3.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD19\u003csup\u003e+\u003c/sup\u003e B cells (cells/\u0026micro;l)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34.1 (50\u0026ndash;670)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e93 (180\u0026ndash;324)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11 (180\u0026ndash;324)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e42 [1.9%]\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e61 [6.20%]\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD56\u003csup\u003e+\u003c/sup\u003e NK cells (cells/\u0026micro;l)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e132.7 (40-1000)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e107 (175\u0026ndash;567)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e46 (175\u0026ndash;567)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e130 [5.9%]\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e244 [14.19%]\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIgA (g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.92 (0.7-4.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5 (0.8-4.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.38 (0.7-4.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.29 (0.7-4.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.34 (0.7-4.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIgG (g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8 (7.0\u0026ndash;16.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.0\u003csup\u003e*\u003c/sup\u003e (7.0\u0026ndash;15.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.15\u003csup\u003e*\u003c/sup\u003e (7.0\u0026ndash;17.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.87 (7.0\u0026ndash;16.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.15\u003csup\u003e*\u003c/sup\u003e (7.0\u0026ndash;16.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIgM (g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.92 (0.4\u0026ndash;2.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.90 (0.4\u0026ndash;2.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.43 (0.4\u0026ndash;2.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.54 (0.4\u0026ndash;2.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.03 (0.4\u0026ndash;2.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD28\u003csup\u003e+\u003c/sup\u003e/CD4\u003csup\u003e+\u003c/sup\u003e (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e58.8 (85.0-100.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD8\u003csup\u003e+\u003c/sup\u003eCD28\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.4 (37.2\u0026ndash;60.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD8\u003csup\u003e+\u003c/sup\u003eHLA-DR\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82.5 (6.3\u0026ndash;23.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD8\u003csup\u003e+\u003c/sup\u003eCD38\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e98.5 (32.4\u0026ndash;57.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eNumbers in parentheses represent reference ranges. \u003csup\u003e*\u003c/sup\u003e Affected by IVIg supplementation. \u003csup\u003e**\u003c/sup\u003e WBC number, proportion of lymphocyte and immune cell subset assay were determined using different samples. \u003csup\u003e\u0026sect;\u003c/sup\u003e Calculated based on subset proportions indicated in brackets. Reference ranges unavailable for subset proportions. \u003csup\u003e#\u003c/sup\u003e Cell numbers calculated based on subset proportions indicated in brackets. Reference ranges for subset proportions: CD3\u003csup\u003e+\u003c/sup\u003e%: 49.1\u0026ndash;83.6%, CD4\u003csup\u003e+\u003c/sup\u003e%: 28.2\u0026ndash;62.8%, CD8\u003csup\u003e+\u003c/sup\u003e%: 10.2\u0026ndash;40.1%, CD19\u003csup\u003e+\u003c/sup\u003e%: 6\u0026ndash;25%, Total NK%: 5\u0026ndash;27%.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFrom November 2018 to March 2019, the patient experienced a deterioration in visual acuity, xanthopsia and distorted vision, first in the right eye and subsequently in the left. A diagnoses of cytomegalovirus (CMV) retinitis was considered and intravitreal injections of ganciclovir were then initiated. However, the visual acuity of both eyes continued to deteriorate. He developed a headache during this period, and a brain MRI revealed neurocysticercosis of the right cerebellar hemisphere. His symptoms improved after the administration of praziquantel. Follow-up His follow-up head MR images are showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC. A hypointensity lesion (indicated with a white arrow) in the cingulate gyrus cortex of the right frontal lobe was demonstrated on both T1-weighted (T1W) and FLAIR images, and no T1W contrast enhancement were observed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince May 2019, the patient experienced recurrent lung infections over the following 6 months. Chest CT images demonstrated diffuse exudation and reticulation in both lungs, particularly in lower lobes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Next-generation sequencing of the bronchoalveolar lavage fluid detected \u003cem\u003eKlebsiella aerogenes\u003c/em\u003e (reads 8), \u003cem\u003ePneumocystis jirovecii\u003c/em\u003e (reads 486) and rubella virus (RV) (reads 158). CMV DNA was detected in peripheral blood (1.29\u0026times;10\u003csup\u003e4\u003c/sup\u003e copies/mL). His eyesight continued to deteriorate due to persistent CMV retinitis. Fundoscopy showed large patches of white granular retinal necrosis lesions and vascular occlusion in both eyes. Ocular ultrasonography showed vitreous opacities and retinal detachment in the right eye (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Next-generation sequencing of the aqueous humor identified CMV (reads 16), RV (reads 3) and \u003cem\u003eStreptococcus equi\u003c/em\u003e (reads 2874). Throughout this period, a series of immunological tests revealed persistent CD4\u003csup\u003e+\u003c/sup\u003e T cell lymphopenia and hypoimmunoglobulinemia (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The clinical possibility of acquired immunodeficiency was considered but later ruled out as tests for anti-IFN-γ autoantibody and HIV RNA were both negative. The lung infection resolved by a combined therapy of meropenem, ganciclovir, SMZ-TMP, caspofungin, and IVIg. Following discharge, the patient remained well for two years, but unfortunately succumbed to an unmanageable central nerve system infection in 2021.\u003c/p\u003e \u003cp\u003eThe patient is from a consanguineous family, with both parents being healthy. His sibling also suffered from recurrent severe lung infections. During her last episode, strong broad-spectrum antimicrobial agents, including cefoperazone/sulbactam, meropenem, linezolid, ganciclovir and voriconazole were used to treat the bacterial (pulmonary consolidation and infiltration lesions on chest CT), CMV (blood CMV DNA 2.06\u0026times;10\u003csup\u003e2\u003c/sup\u003e copies) and suspected fungal (positive results of blood G test at different time points) infections in the lungs. Despite the implementation of IVIg and mechanical ventilation, she kept deteriorating, and eventually died of severe respiratory infection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eImmunological features of the patient\u003c/h2\u003e \u003cp\u003eDuring the course of disease, he showed a persistent deficient in both humoral and cellular immunity. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, serial immunological tests conducted during 2018\u0026ndash;2019 revealed that the patient exhibited persistently reduced numbers of CD4\u003csup\u003e+\u003c/sup\u003e T and B cells, profound hypoimmunoglobulinemia, and sometimes a decreased number of NK cells. A similar pattern of T-cell lymphopenia (CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e/CD45\u003csup\u003e+\u003c/sup\u003e 14.8%, reference range: 30.0\u0026ndash;54.0%) and hypoimmunoglobulinemia (IgG \u0026lt;0.333 g/L, reference range: 7.51\u0026ndash;15.60 g/L; IgA 0.16 g/L, reference range: 0.82\u0026ndash;4.53 g/L; IgM 0.10 g/L, reference range: 0.46\u0026ndash;3.04 g/L) was also observed in his sister. The patient showed increased number of CD8\u003csup\u003e+\u003c/sup\u003e T cells, which showed a more activated and differentiated phenotype as indicated by an elevated proportion of HLA-DR\u003csup\u003e+\u003c/sup\u003e or CD38\u003csup\u003e+\u003c/sup\u003e cells(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Both the CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T-cell subsets displayed a significantly lower proportion of CD28\u003csup\u003e+\u003c/sup\u003e cells, suggesting a decrease in na\u0026iuml;ve T cell which express a high level of differentiation marker CD28(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Due to the limited availability of samples collected prior to the patient\u0026rsquo;s passing, further detailed immunological analysis of the patient could not be conducted.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of a novel germline missense mutation in\u003c/b\u003e \u003cb\u003eRHOH\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe patient\u0026rsquo;s parents were consanguineous cousins, and both were in good health. However, the patient\u0026rsquo;s elder sister died at her 20s due to uncontrolled lung infection. Based on the patient\u0026rsquo;s clinical characteristics and family history, autosomal recessive immunologic disorders were suspected. Whole-exome sequencing of the kindred was therefore performed. We identified a total of 15 nonsynonymous single nucleotide variants (\u003cem\u003ePRF1, HELLS, LRP1, PAH, BLM, RLTPR, CD226, LIG1, ALK, ADA, RHOH, EGFR, MET, DDX58, PTCH1\u003c/em\u003e) and 2 frameshift deletions (\u003cem\u003eMICA, BTNL2\u003c/em\u003e) in the exons of protein-coding genes with a minor allele frequency lower than 0.01 in population databases, including 1000 Genomes Project database, dbSNP, Exome Aggregation Consortium or Genome Aggregation Database. Among these rare variants, the variant in \u003cem\u003eRHOH\u003c/em\u003e is the only homozygous one in the patient. Both parents are heterozygous carriers of this variant, supporting an autosomal-recessive inheritance pattern for this novel germline \u003cem\u003eRHOH\u003c/em\u003e mutation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The variant (GenBank: NM_004310.5: c.245G\u0026thinsp;\u0026gt;\u0026thinsp;A, p.Cys82Tyr) resulted in substitution of an evolutionary conserved cysteine residue with a tyrosine residue in the ITAM-like domain responsible for interaction with ZAP70 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The C82Y \u003cem\u003eRHOH\u003c/em\u003e variant has not been reported in population databases. It is predicted to be pathogenic by multiple in silico pathogenicity prediction tools, including CADD (score\u0026thinsp;=\u0026thinsp;26.4), REVEL (0.831, pathogenic), SIFT (0.001, deleterious), PolyPhen-2 (1.0, probably damaging), and MutationTaster (1, disease causing). Unfortunately, genetic assays of the patient\u0026rsquo;s sibling could not be performed due to the unavailability of a specimen. HPV infection was found in previous reported \u003cem\u003eRHOH\u003c/em\u003e deficiency cases, but HPV serology test results of our patient and his parents were negative.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eExpression and function of RHOH\u003csup\u003eC82Y\u003c/sup\u003e\u003c/h2\u003e \u003cp\u003eWe first assessed the effect of the C82Y variant on the mRNA expression of \u003cem\u003eRHOH\u003c/em\u003e gene using quantitative-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The results showed that \u003cem\u003eRHOH\u003c/em\u003e mRNA expression in peripheral blood was comparable to that of his parents and the controls. An impaired T cell activation has been reported in previous \u003cem\u003eRHOH\u003c/em\u003e deficiency cases (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), we therefore analyzed how T cells respond to anti-CD3/CD28 stimulation in these kindred. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, PBMC from the patient contained a much lower proportion of viable cells than did his parents, with or without TCR stimulation. The patient\u0026rsquo;s T cells failed to enlarge, as indicated by the significantly lower FSC of viable cells compared to his parents, suggesting a general defect in TCR-mediated T cell activation in the patient.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDetailed immunologic analysis of the patient could not be done, because the patient succumbed to infection, and no more biological specimens were available for investigation. We therefore evaluate the expression and function of the C82Y RHOH variant using cell lines generated to express RHOH\u003csup\u003eWT\u003c/sup\u003e or RHOH\u003csup\u003eC82Y\u003c/sup\u003e. Comparable levels of wild type and mutant RHOH protein were observed in HEK293 cell line. However, the ectopic expression of HA-tagged RHOH in Jurkat cells revealed significantly compromised protein expression by the C82Y variant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eThe C82Y variant maps to the ITAM domain of RHOH which interacts with ZAP70, a key molecule in TCR signaling pathway (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).To assess whether the C82Y variant disrupt the interaction between RHOH and ZAP70, co-immunoprecipitation was performed using Jurkat cells lysate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). In RHOH\u003csup\u003eWT\u003c/sup\u003e-expressing Jurkat cells, ZAP70 could bind with RHOH in a resting state, and RHOH-bound ZAP70 increased after anti-CD3 stimulation, consistent with previous publications(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Meanwhile, in Jurkat cells ectopically expressing RHOH\u003csup\u003eC82Y\u003c/sup\u003e, the protein expression level of the C82Y variant was significantly lower than that of the wild type, even after enrichment. More importantly, the binding between ZAP70 and RHOH was undetectable, both before and after anti-CD3 stimulation. Therefore, our findings demonstrate that the C82Y variant impairs RHOH function by undermining its interaction with downstream signaling molecule ZAP70.\u003c/p\u003e \u003cp\u003eTo verify whether the C82Y variant disrupted the signal transduction of TCR, we compared the expression of T-cell activation marker CD69 (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) in Jurkat cells ectopically expressing RHOH\u003csup\u003eWT\u003c/sup\u003e, the C82Y variant, or an empty vector control (EV) upon anti-CD3 stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). In comparison to EV, a rapid elevation of CD69 level was observed in Jurkat cells expressing RHOH\u003csup\u003eWT\u003c/sup\u003e. In contrast, the expression of CD69 in RHOH\u003csup\u003eC82Y\u003c/sup\u003e-expressing Jurkat cells failed to up-regulate, and remained at a level comparable to that of EV Jurkat cells. These observations suggest that the C82Y is a loss-of-function variant but does not exert a dominant negative effect over the endogenous WT RHOH. Measurement of IL-2 production revealed similar results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). The culture supernatants of Jurkat cells expressing RHOH\u003csup\u003eC82Y\u003c/sup\u003e contained a much lower level of IL-2 than that of RHOH\u003csup\u003eWT\u003c/sup\u003e-expressing Jurkat cells. Collectively, these results indicate that the C82Y variant is a hypomorphic mutation which hampers RHOH protein expression and impairs its function in TCR-mediated T cell activation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe \u003cem\u003eRHOH\u003c/em\u003e gene encodes a hematopoietic-specific Rho GTPase expressed in myeloid, NK, B, and T lymphocytes. As a membrane-bound adaptor for proximal protein kinases involved in TCR signal transduction, \u003cem\u003eRHOH\u003c/em\u003e exerts a critical role in the activation, development and differentiation of T lymphocytes (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Somatic mutations in \u003cem\u003eRHOH\u003c/em\u003e gene have been linked to certain hematological malignancies (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). A germline mutation in \u003cem\u003eRHOH\u003c/em\u003e has been identified as the cause of an autosomal recessive IEI (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). To date, only one kindred of \u003cem\u003eRHOH\u003c/em\u003e deficiency, also called epidermodysplasia verruciformis-4, has been documented (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). The probands are two siblings from a consanguineous family. Both had a homozygous Y38X nonsense variant in \u003cem\u003eRHOH\u003c/em\u003e gene. The Y38X variant led to a complete loss of RHOH protein expression. Both patients experienced early-onset disease. The proband, since the age of 5, developed Burkitt lymphoma, epidermodysplasia verruciformis, skin lesions due to HPV subtypes 3, 12, and 20, as well as pulmonary diseases, including a granulomatous lesion, chronic bronchial disease with emphysema, and right pneumothorax. His sister suffered from gingivostomatitis and similar treatment-resistant skin lesions caused by HPV subtype 20 from the age of 2.\u003c/p\u003e \u003cp\u003eOur case presents a distinct clinical phenotype. Firstly, our patient experienced a late disease onset at the age of 21 years. Secondly, the typical skin lesion associated with epidermodysplasia verruciformis was absent, and no evidence of HPV infection was found. Thirdly, the patient exhibited recurrent infections affecting multiple organs, including the lungs, retina, and brain, caused by various opportunistic pathogens such as CMV, RV, \u003cem\u003eP. jirovecii\u003c/em\u003e, helminth, and multiple bacteria. Similarly, his sibling also suffered from recurrent severe lung infections and free from HPV-related skin conditions.\u003c/p\u003e \u003cp\u003eOur patient\u0026rsquo;s immunophenotype shared similarities with previously reported \u003cem\u003eRHOH\u003c/em\u003e deficiency cases in terms of reduced CD4\u003csup\u003e+\u003c/sup\u003e T cells, increased CD8\u003csup\u003e+\u003c/sup\u003e T cells, and an elevated proportion of activated, differentiated effector and memory CD8\u003csup\u003e+\u003c/sup\u003e T cells. Crequer, A. \u003cem\u003eet al.\u003c/em\u003e revealed that the RHOH\u003csup\u003eY38X\u003c/sup\u003e variant resulted in a complete absence of \u003cem\u003eRHOH\u003c/em\u003e protein expression, defects in T-cell development and TCR signaling, impaired T-cell proliferation in response to certain antigens, and tissue-homing defects (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). In our case, the C82Y variant allows for normal transcription of the \u003cem\u003eRHOH\u003c/em\u003e mutant, but disrupted its protein expression. The impaired response to TCR stimulation of the patient T cell was consistent with previous \u003cem\u003eRHOH\u003c/em\u003e deficiency cases. The defective upregulation of CD69 and production of IL-2 in RHOH\u003csup\u003eC82Y\u003c/sup\u003e-expressing Jurkat cells also support that the C82Y variant is functionally impaired, particularly in TCR-induced T cell activation.\u003c/p\u003e \u003cp\u003eStudies on \u003cem\u003eRhoH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice have observed a defect in thymocyte selection, maturation, lower T-cell cytotoxicity and cytokine release, and have demonstrated that it is a result of reduced activation of ZAP70-mediated signaling and impaired translocation of ZAP70 to the immunological synapse (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). The function of RHOH depends on the phosphorylation of its ITAMs (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Considering that the C82Y variant is located within the ITAM-like motif, it is plausible that the defective function of the RHOH\u003csup\u003eC82Y\u003c/sup\u003e variant arises from its impaired interaction with ZAP70. Results from the immunoprecipitation analysis support our hypothesis, as RHOH-bound ZAP70 was undetectable in RHOH\u003csup\u003eC82Y\u003c/sup\u003e Jurkat cells. The C82Y variant led to a major defect in the interaction between RHOH and ZAP70, and impaired subsequent TCR signaling, at an extent similarly to what has been observed in cases with stop-gain \u003cem\u003eRHOH\u003c/em\u003e mutation. Nevertheless, Dorn, T., \u003cem\u003eet al.\u003c/em\u003e found that RHOH is not required for TCR-induced activation of ZAP70, but is indispensable for the efficient interaction of ZAP70 with the LAT signalosome (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Since RHOH-bound ZAP70 was barely detected, we were unable to determine the phosphorylation and activation of ZAP70. Previous research also proposed that RHOH regulates T-cell development and function by maintaining LCK in an inactivated states (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), allowing T cells to switch between sensing chemokine-mediated go signals and TCR-dependent stop signals (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), and maintaining lymphocyte LFA-1 in a nonadhesive state (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). It also played a role in the differentiation of Th\u003csub\u003e17\u003c/sub\u003e cell by modulating the expression and function of RORγt (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Our data provide an explanation for how the RHOH\u003csup\u003eC82Y\u003c/sup\u003e variant might be responsible for the T cell deficiency in this patient. But, further investigation is required, to understand the role of RHOH\u003csup\u003eC82Y\u003c/sup\u003e mutant in pre-TCR, TCR signaling and regulation of T-cell function.\u003c/p\u003e \u003cp\u003eOur case also exhibited significant differences in the immunophenotype. In the previously reported cases, no obvious abnormalities were observed in B cells, NK cells, NKT cells, or antibody production. In contrast, our patient presented a persistent decrease in B and NK cell numbers, and profound hypoimmunoglobulinemia, indicating a combined defect in both cellular and humoral immune responses. This immunophenotype aligns with the expression pattern of \u003cem\u003eRHOH\u003c/em\u003e in immune cells. The RHOH\u003csup\u003eC82Y\u003c/sup\u003e might also be associated with abnormality in BCR signaling, However, BCR signaling has also been found intact in \u003cem\u003eRhoh\u003c/em\u003e-deficient mice (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). A recent study in patients with CD28 deficiency strongly suggest that the poor antibody responses seen in those patients after vaccination results from a deficiency of CD4 T cell response, due to the absence of CD28 co-stimulation signaling (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). In order to provide an explanation for the pathogenesis of B and NK cell deficiency in our patient with the RHOH\u003csup\u003eC82Y\u003c/sup\u003e variant, further investigation using mouse model is required.\u003c/p\u003e \u003cp\u003eIn this study, we reported a novel homozygous germline variant in the \u003cem\u003eRHOH\u003c/em\u003e gene (p.Cys82Tyr), associated with an autosomal recessive IEI. The affected individual exhibited recurrent, invasive, opportunistic infections in the lungs, eyes and brain during early adulthood, accompanied by a persistent decrease in CD4\u003csup\u003e+\u003c/sup\u003e T, B, NK cells, and hypoimmunoglobulinemia. Immunological analysis demonstrated both reduced protein expression and defective binding of the C82Y mutant to the signaling molecule ZAP70. Impaired TCR-mediated T-cell activation was observed in both the patient and in RHOH\u003csup\u003eC82Y\u003c/sup\u003e-expressing Jurkat cells, indicating that the C82Y variant is a hypomorph associated with abnormalities in TCR signaling. Moreover, we confirmed that the C82Y variant does not exert a dominant negative effect. In conclusion, our case expands the genetic and phenotypic spectrum of \u003cem\u003eRHOH\u003c/em\u003e deficiency and provides new insights into the development and regulation of T, B and NK cells.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (grant #82271794 to Q-L R and grants\u0026nbsp;#32330033 and\u0026nbsp;#32270932 to J-YW).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.Y. Zhou, M.Q. Qian\u0026nbsp;participated in\u0026nbsp;research investigation, data analysis and writing-original draft; J. Ning and J. Wu\u0026nbsp;participated in\u0026nbsp;research investigation,\u0026nbsp;data analysis,\u0026nbsp;contributed to methodology and software, writing-review \u0026amp; editing; X.Q. Feng, M.P. Yu and M. Qin\u0026nbsp;participated in\u0026nbsp;research investigation,\u0026nbsp;contributed to methodology; H.X. Xu, Y.X. Yang, Q.L. Yang, F.R. Zhou, L.Y. Shao, H.X. Zhu and Y. Yang participated in clinical management and data collection of the kindred; Q.L. Ruan and J.Y. Wang:\u0026nbsp;participated in research design, writing-editing and research funding, contributed research materials; W.H. Zhang participated in project administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe exome sequencing data are not publicly available due to privacy restrictions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study received approval from the institutional ethics review board of Huashan Hospital, Fudan University, and was conducted in accordance with the principles outlined in the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGu Y, Jasti AC, Jansen M, Siefring JE. RhoH, a hematopoietic-specific Rho GTPase, regulates proliferation, survival, migration, and engraftment of hematopoietic progenitor cells. Blood. 2005;105(4):1467-75.\u003c/li\u003e\n\u003cli\u003eSchmidt-Mende J, Geering B, Yousefi S, Simon HU. Lysosomal degradation of RhoH protein upon antigen receptor activation in T but not B cells. Eur J Immunol. 2010;40(2):525-9.\u003c/li\u003e\n\u003cli\u003eStoeckle C, Geering B, Yousefi S, Rožman S, Andina N, Benarafa C, et al. RhoH is a negative regulator of eosinophilopoiesis. Cell Death Differ. 2016;23(12):1961-72.\u003c/li\u003e\n\u003cli\u003eAhmad Mokhtar AM, Hashim IF, Mohd Zaini Makhtar M, Salikin NH, Amin-Nordin S. The Role of RhoH in TCR Signalling and Its Involvement in Diseases. Cells. 2021;10(4).\u003c/li\u003e\n\u003cli\u003eGu Y, Chae HD, Siefring JE, Jasti AC, Hildeman DA, Williams DA. RhoH GTPase recruits and activates Zap70 required for T cell receptor signaling and thymocyte development. Nat Immunol. 2006;7(11):1182-90.\u003c/li\u003e\n\u003cli\u003eCrequer A, Troeger A, Patin E, Ma CS, Picard C, Pedergnana V, et al. Human RHOH deficiency causes T cell defects and susceptibility to EV-HPV infections. J Clin Invest. 2012;122(9):3239-47.\u003c/li\u003e\n\u003cli\u003eMahnke YD, Brodie TM, Sallusto F, Roederer M, Lugli E. The who\u0026apos;s who of T-cell differentiation: human memory T-cell subsets. Eur J Immunol. 2013;43(11):2797-809.\u003c/li\u003e\n\u003cli\u003eZiegler SF, Ramsdell F, Alderson MR. The activation antigen CD69. Stem Cells. 1994;12(5):456-65.\u003c/li\u003e\n\u003cli\u003eChae HD, Siefring JE, Hildeman DA, Gu Y, Williams DA. RhoH regulates subcellular localization of ZAP-70 and Lck in T cell receptor signaling. PLoS One. 2010;5(11):e13970.\u003c/li\u003e\n\u003cli\u003eDorn T, Kuhn U, Bungartz G, Stiller S, Bauer M, Ellwart J, et al. RhoH is important for positive thymocyte selection and T-cell receptor signaling. Blood. 2007;109(6):2346-55.\u003c/li\u003e\n\u003cli\u003eWang H, Zeng X, Fan Z, Lim B. RhoH modulates pre-TCR and TCR signalling by regulating LCK. Cell Signal. 2011;23(1):249-58.\u003c/li\u003e\n\u003cli\u003eBaker CM, Comrie WA, Hyun YM, Chung HL, Fedorchuk CA, Lim K, et al. Opposing roles for RhoH GTPase during T-cell migration and activation. Proc Natl Acad Sci U S A. 2012;109(26):10474-9.\u003c/li\u003e\n\u003cli\u003eCherry LK, Li X, Schwab P, Lim B, Klickstein LB. RhoH is required to maintain the integrin LFA-1 in a nonadhesive state on lymphocytes. Nat Immunol. 2004;5(9):961-7.\u003c/li\u003e\n\u003cli\u003eTamehiro N, Nishida K, Sugita Y, Hayakawa K, Oda H, Nitta T, et al. Ras homolog gene family H (RhoH) deficiency induces psoriasis-like chronic dermatitis by promoting T(H)17 cell polarization. J Allergy Clin Immunol. 2019;143(5):1878-91.\u003c/li\u003e\n\u003cli\u003eSanchez-Aguilera A, Rattmann I, Drew DZ, M\u0026uuml;ller LU, Summey V, Lucas DM, et al. Involvement of RhoH GTPase in the development of B-cell chronic lymphocytic leukemia. Leukemia. 2010;24(1):97-104.\u003c/li\u003e\n\u003cli\u003eB\u0026eacute;ziat V, Rapaport F, Hu J, Titeux M, Bonnet des Claustres M, Bourgey M, et al. Humans with inherited T cell CD28 deficiency are susceptible to skin papillomaviruses but are otherwise healthy. Cell. 2021;184(14):3812-28.e30.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-clinical-immunology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joci","sideBox":"Learn more about [Journal of Clinical Immunology](https://www.springer.com/journal/10875)","snPcode":"10875","submissionUrl":"https://submission.nature.com/new-submission/10875/3","title":"Journal of Clinical Immunology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"RHOH, T cell, TCR signaling, opportunistic infection, primary immunodeficiency","lastPublishedDoi":"10.21203/rs.3.rs-3958385/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3958385/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRHOH, an atypical small GTPase predominantly expressed in hematopoietic cells, plays a vital role in immune function. A deficiency in RHOH has been linked to epidermodysplasia verruciformis, lung disease, Burkitt lymphoma and T cell defects. Here, we report a novel germline homozygous \u003cem\u003eRHOH\u003c/em\u003e c.245G\u0026thinsp;\u0026gt;\u0026thinsp;A (p.Cys82Tyr) variant in a 21-year-old male suffering from recurrent, invasive, opportunistic infections affecting the lungs, eyes, and brain. His sister also succumbed to a lung infection during early adulthood. The patient exhibited a persistent decrease in CD4\u003csup\u003e+\u003c/sup\u003e T, B, and NK cell counts, and hypoimmunoglobulinemia. Despite normal \u003cem\u003eRHOH\u003c/em\u003e mRNA levels in his family, the patient\u0026rsquo;s T cell showed impaired activation upon in vitro TCR stimulation. In Jurkat T cells transduced with RHOH\u003csup\u003eC82Y\u003c/sup\u003e, a similar reduction in CD69 activation marker up-regulation was observed. However, ectopic expression of the C82Y variant did not exhibit a negative dominance over wild type RHOH. Furthermore, the C82Y variant showed reduced RHOH protein expression and impaired interaction with the TCR signaling molecule ZAP70. Together, these data suggest that the newly identified autosomal-recessive \u003cem\u003eRHOH\u003c/em\u003e variant is associated with T cell dysfunction and recurrent opportunistic infections, functioning as a hypomorph by disrupting ZAP70-mediated TCR signaling.\u003c/p\u003e","manuscriptTitle":"A novel homozygous RHOH variant associated with T cell dysfunction and recurrent opportunistic infections","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-21 18:02:28","doi":"10.21203/rs.3.rs-3958385/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-29T00:25:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-04T12:51:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5b84ce8d-2ea7-480a-beee-40f632d464be","date":"2024-02-25T05:46:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-23T01:30:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-19T07:14:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-19T07:14:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Clinical Immunology","date":"2024-02-15T10:33:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-clinical-immunology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joci","sideBox":"Learn more about [Journal of Clinical Immunology](https://www.springer.com/journal/10875)","snPcode":"10875","submissionUrl":"https://submission.nature.com/new-submission/10875/3","title":"Journal of Clinical Immunology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d05fd7b4-2c39-4bf1-b3c3-bda0d15fe1df","owner":[],"postedDate":"February 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-06-28T08:15:52+00:00","versionOfRecord":{"articleIdentity":"rs-3958385","link":"https://doi.org/10.1007/s10875-024-01735-4","journal":{"identity":"journal-of-clinical-immunology","isVorOnly":false,"title":"Journal of Clinical Immunology"},"publishedOn":"2024-05-22 08:15:52","publishedOnDateReadable":"May 22nd, 2024"},"versionCreatedAt":"2024-02-21 18:02:28","video":"","vorDoi":"10.1007/s10875-024-01735-4","vorDoiUrl":"https://doi.org/10.1007/s10875-024-01735-4","workflowStages":[]},"version":"v1","identity":"rs-3958385","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3958385","identity":"rs-3958385","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}