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A monoallelic activating mutation in RAC2 resulting in a combined immunodeficiency

2019; Elsevier BV; Volume: 143; Issue: 4 Linguagem: Inglês

10.1016/j.jaci.2019.01.001

1097-6825

Vassilios Lougaris, Janet Chou, Abdallah Beano, Jacqueline G. Wallace, Manuela Baronio, Luisa Gazzurelli, Tiziana Lorenzini, Daniele Moratto, Giovanna Tabellini, Silvia Parolini, Michael Seleman, Kelsey Stafstrom, Haiming Xu, Chad E. Harris, Raif S. Geha, Alessandro Plebani,

Immunodeficiency and Autoimmune Disorders

The Rho guanosine triphosphatases, RAC1, RAC2, and RAC3, regulate cellular functions by switching from the inactive guanosine diphosphate–bound state to the active guanosine triphosphate–bound state.1Pai S.-Y. Kim C. Williams D.A. Rac GTPases in human diseases.Dis Markers. 2010; 29: 177-187Crossref PubMed Google Scholar RAC1 and RAC3 are widely expressed, whereas RAC2 is expressed only in hematopoietic cells.1Pai S.-Y. Kim C. Williams D.A. Rac GTPases in human diseases.Dis Markers. 2010; 29: 177-187Crossref PubMed Google Scholar The mouse model of RAC2 deficiency and patients with the dominant negative RAC2D57N mutant have neutrophilia, recurrent abscesses, and defective wound healing.2Ambruso D.R. Knall C. Abell A.N. Panepinto J. Kurkchubasche A. Thurman G. et al.Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation.Proc Natl Acad Sci U S A. 2000; 97: 4654-4659Crossref PubMed Scopus (362) Google Scholar, 3Williams D.A. Tao W. Yang F. Kim C. Gu Y. Mansfield P. et al.Dominant negative mutation of the hematopoietic-specific Rho GTPase, Rac2, is associated with a human phagocyte immunodeficiency.Blood. 2000; 96: 1646-1654PubMed Google Scholar Because of the contribution of RAC2 to the function of the glyceraldehyde-3-phosphate dehydrogenase oxidase complex, RAC2D57N impairs the generation of superoxide in neutrophils. RAC2D57N also disrupts the polymerization of filamentous actin, thereby reducing myeloid cell and lymphocyte chemotaxis and adhesion.2Ambruso D.R. Knall C. Abell A.N. Panepinto J. Kurkchubasche A. Thurman G. et al.Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation.Proc Natl Acad Sci U S A. 2000; 97: 4654-4659Crossref PubMed Scopus (362) Google Scholar, 3Williams D.A. Tao W. Yang F. Kim C. Gu Y. Mansfield P. et al.Dominant negative mutation of the hematopoietic-specific Rho GTPase, Rac2, is associated with a human phagocyte immunodeficiency.Blood. 2000; 96: 1646-1654PubMed Google Scholar Although conditional deletion of either RAC1 or RAC2 in lymphocytes is sufficient for intact T-cell and B-cell development in mice, reduced numbers of CD4+ T cells and B cells have been found in one patient with RAC2D57N and in another kindred lacking RAC2 protein expression due to a homozygous RAC2W56X mutation.4Alkhairy O.K. Rezaei N. Graham R.R. Abolhassani H. Borte S. Hultenby K. et al.RAC2 loss-of-function mutation in 2 siblings with characteristics of common variable immunodeficiency.J Allergy Clin Immunol. 2015; 135: 1380-1384.e5Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 5Accetta D. Syverson G. Bonacci B. Reddy S. Bengtson C. Surfus J. et al.Human phagocyte defect caused by a Rac2 mutation detected by means of neonatal screening for T-cell lymphopenia.J Allergy Clin Immunol. 2011; 127 (e1-2): 535-538Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar Somatic activating variants in RAC2 have been associated with melanoma,1Pai S.-Y. Kim C. Williams D.A. Rac GTPases in human diseases.Dis Markers. 2010; 29: 177-187Crossref PubMed Google Scholar but there are no published reports of germline gain-of-function mutations in any RAC family members. Here, we report a monoallelic germline gain-of-function RAC2P34H mutant in a family with a combined immunodeficiency. Patient 1 is a 39-year-old man with a history of bronchiectasis, recurrent sinopulmonary infections, cutaneous human papillomavirus infections, and basal cell carcinomas. At age 34 years, he developed antiphospholipid syndrome and an IgM monoclonal gammopathy in association with a low-grade B-cell non-Hodgkin lymphoma, which was treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone. His daughters (patients 2 and 3; Fig 1, A) had a history of recurrent sinopulmonary infections, severe lymphopenia since early childhood, and mild intermittent neutropenia (see Fig E1, A, in this article's Online Repository at www.jacionline.org). All patients are undergoing treatment with immunoglobulin replacement and antibiotic prophylaxis. The patients had reduced CD4+ and CD8+ T cells, recent thymic emigrants, naive T cells, and proliferative responses to stimulation with mitogens (Table I). All 3 patients had an accumulation of senescent CD8+CD57+ T cells (Table I). The patients had low CD19+ B cells. Immunoglobulin levels before initiation of immunoglobulin replacement were available for patients 2 and 3. They demonstrated hypogammaglobulinemia and undetectable titers to tetanus and hepatitis vaccines (Table I). Collectively, these findings indicate a combined immunodeficiency.Table IImmune phenotyping of the patients' lymphocytesVariablePatient 1Patient 2Patient 3Age at the time of testing (y)3172Hemogram (normal range) Hemoglobin (g/dL)15.6 (10.9-15)12.6 (10.5-13.8)11.8 (11-12.8) WBCs (103 cells/μL)3.670 (5.8-9.3)1.640 (5.4-9.7)2.960 (5.9-10.4) Neutrophils (103 cells/μL)2.822 (3.3-6.3)0.730 (2.5-5.9)1.557 (2.5-6) Lymphocytes (103 cells/μL)0.499 (1.14-2.28)0.350 (1.28-2.76)0.400 (1.33-4.77) Monocytes (103 cells/μL)0.184 (0.29-0.7)0.172 (0.19-0.81)0.266 (0.19-0.94) Platelets (103 cells/μL)153 (174-333)214 (187-367)216 (208-413)Lymphocyte subsets CD3+ (103 cells/μL)359 (1000-2600)248 (1400-3700)252 (2100-6200)CD3+CD4+ (103 cells/μL)72 (530-1500)140 (700-2200)98 (1300-3400)CD45RA+CD31+ recent thymic emigrants, % CD4+0.0 (9.8-43)32 (45-63)29 (57-76)CD45RA+CCR7+CD31– naive, % CD4+0.7 (21-61)36.4 (57.1-84.8)32.7 (65-84)CD45RA–CCR7– effector memory, % CD4+95.8 (7.6-25)44.1 (3.3-15.2)35.7 (2.9-9.8)CD45RA–CCR7+ central memory, % CD4+3.3 (26-62)16.9 (11.2-26.7)29.8 (10-22.3)CD3+CD8+ (103 cells/μL)224 (330-1100)55 (490-1300)65 (620-2000)CD45RA+CCR7+CD31– naive, % CD8+0.4 (11-66)4.6 (28-80)14.7 (39-89)CD45RA–CCR7– effector memory, % CD8+60.9 (16-54)56.2 (6.2-29.3)36.4 (3.4-28)CD45RA–CCR7+ central memory, % CD8+0.8 (3.7-32)1.2 (1.2-4.5)6.3 (1-5.7)CD45RA+CCR7− TEMRA, % CD8+38.1 (5.6-43)38 (9.1-49.1)42.7 (4.8-30)CD57+, % CD8+∗Daball et al.667.9 (2-38)50 (2-20)42.8 (2-16) CD19+ (103 cells/μL)12 (110-570)40 (390-1400)19 (720-2600)CD27−IgD+IgM+ naive, % CD19+60.8 (48-97)30.2 (47-77)25.0 (54-88)CD27+IgD+ unswitched memory, % CD19+27.4 (7-23)8.8 (5.2-20.4)11.9 (2.7-19)CD27+IgD– switched memory, % CD19+9.4 (8.8-27)21.7 (10.9-30.4)19.6 (3.3-7.4)CD38hiIgMhitransitional, % CD19+0.8 (2.2-13)35.6 (7.2-23)37.8 (10-30) CD3–CD56+ (103 cells/μL)101 (70-480)56 (130-0.720)116 (160-920)Immunoglobulins IgG (mg/dL)804†On IVIG. (639-1349)353 (463-1236)239 (345-1213) IgM (mg/dL)83 (56-352)14 (43-196)12 (14-106) IgA (mg/dL)226 (70-312)17 (25-154)13 (43-173) Tetanus vaccine titern.d. before IVIG startedUDUD Hepatitis vaccine titern.d. before IVIG startedUDUDProliferation‡Reference range is for 3 healthy controls done on the day of the study. Anti-CD3 (% CD4+ divided)65.3 (84.2-93.8)89.05 (84.2-93.8)24.5 (84.2-93.8) Phytohemmaglutinin (% CD4+ divided)6.04 (62.9-85)60 (62.9-85)63.85 (62.9-85) Anti-CD3 (% CD8+ divided)54.5 (87-92.7)64.45 (87-92.7)34.3 (87-92.7) Phytohemmaglutinin (% CD8+ divided)3.81 (83.1-93.5)21.6 (83.1-93.5)20.75 (83.1-93.5)IVIG, Intravenous immunoglobulin; n.d., not done; UD, undetectable; WBC, white blood cell.Boldface values are outside the normal range.∗ Daball et al.6Cura Daball P. Ventura Ferreira M.S. Ammann S. Klemann C. Lorenz M.R. Warthorst U. et al.CD57 identifies T cells with functional senescence before terminal differentiation and relative telomere shortening in patients with activated PI3 kinase delta syndrome.Immunol Cell Biol. 2018; 96: 1060-1071Crossref PubMed Scopus (12) Google Scholar† On IVIG.‡ Reference range is for 3 healthy controls done on the day of the study. Open table in a new tab IVIG, Intravenous immunoglobulin; n.d., not done; UD, undetectable; WBC, white blood cell. Boldface values are outside the normal range. Whole-exome sequencing on all 3 patients identified 51 rare deleterious variants shared by all 3 patients (see Table E1 in this article's Online Repository at www.jacionline.org). Of these, a heterozygous missense mutation in RAC2 (NM_002872: c.C101A; p.P34H) was considered a plausible candidate due to the importance of RAC2 in immune cell function. The mutation was confirmed by Sanger sequencing (Fig 1, B) and had a deleterious Combined Annotation Dependent Depletion Phred-like score of 32.7Kircher M. Witten D.M. Jain P. O'Roak B.J. Cooper G.M. Shendure J. A general framework for estimating the relative pathogenicity of human genetic variants.Nat Genet. 2014; 46: 310-315Crossref PubMed Scopus (3707) Google Scholar The mutated P34 residue resides within the highly conserved Switch 1 domain important for interactions with guanine exchange factors and downstream effectors (Fig 1, C).1Pai S.-Y. Kim C. Williams D.A. Rac GTPases in human diseases.Dis Markers. 2010; 29: 177-187Crossref PubMed Google Scholar Immunoblotting revealed similar levels of RAC2 in patients and controls (Fig E1, B). Because the patients' primary cells express both wild-type (WT) and RAC2P34H, we expressed Flag-tagged WT or mutant RAC2 in HEK293T cells and confirmed that WT and mutant forms of RAC2 had comparable expression (Fig 1, D). Modeling of RAC2P34H suggested a potential interaction between guanosine triphosphate and P34H, which would stabilize the binding of active RAC2 to effector proteins (Fig 1, E). RAC2P34H had increased binding to the effector protein PAK compared with WT RAC2, which was reversed by loading with endogenous guanosine diphosphate (Fig 1, F). RAC2P34H therefore has a gain-of-function effect. RAC2 regulates the activity of the actin depolymerizing protein cofilin.1Pai S.-Y. Kim C. Williams D.A. Rac GTPases in human diseases.Dis Markers. 2010; 29: 177-187Crossref PubMed Google Scholar Dominant negative or loss-of-function mutations in RAC2 result in reduced actin polymerization, chemotaxis, and neutrophil oxidative burst after N-formylmethionyl-leucyl-phenylalanine stimulation.2Ambruso D.R. Knall C. Abell A.N. Panepinto J. Kurkchubasche A. Thurman G. et al.Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation.Proc Natl Acad Sci U S A. 2000; 97: 4654-4659Crossref PubMed Scopus (362) Google Scholar, 3Williams D.A. Tao W. Yang F. Kim C. Gu Y. Mansfield P. et al.Dominant negative mutation of the hematopoietic-specific Rho GTPase, Rac2, is associated with a human phagocyte immunodeficiency.Blood. 2000; 96: 1646-1654PubMed Google Scholar In contrast, the patients' neutrophils demonstrated increased filamentous actin content and an increased respiratory burst after N-formylmethionyl-leucyl-phenylalanine stimulation compared with controls (Fig 1, G and H). The patients' cells had a normal oxidative burst at baseline and after phorbol 12-myristate 13-acetate stimulation, both of which are independent of RAC2 (Fig 1, H).1Pai S.-Y. Kim C. Williams D.A. Rac GTPases in human diseases.Dis Markers. 2010; 29: 177-187Crossref PubMed Google Scholar, 2Ambruso D.R. Knall C. Abell A.N. Panepinto J. Kurkchubasche A. Thurman G. et al.Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation.Proc Natl Acad Sci U S A. 2000; 97: 4654-4659Crossref PubMed Scopus (362) Google Scholar, 3Williams D.A. Tao W. Yang F. Kim C. Gu Y. Mansfield P. et al.Dominant negative mutation of the hematopoietic-specific Rho GTPase, Rac2, is associated with a human phagocyte immunodeficiency.Blood. 2000; 96: 1646-1654PubMed Google Scholar Transgenic mice expressing the constitutively active RAC2G12V or RAC2Q61L mutants demonstrate thymocyte and T-cell apoptosis.1Pai S.-Y. Kim C. Williams D.A. Rac GTPases in human diseases.Dis Markers. 2010; 29: 177-187Crossref PubMed Google Scholar Our patients had lymphopenia with a paucity of naive T cells, reduced recent thymic emigrants, and an accumulation of senescent CD8+CD57+ T cells (Table I). The patients' unstimulated and TCR-stimulated T cells demonstrated increased apoptosis in vitro (Fig. 1, I). In addition, EBV-transformed B cells available from patients 2 and 3 demonstrated decreased viability after serum starvation (Fig E1, C), mirroring their in vivo B-cell lymphopenia. Several mechanisms may contribute to the impaired survival of the patients' T and B cells. Filamentous actin accumulation is known to reduce mitochondrial membrane potential, increase reactive oxidant species, and trigger proapoptotic pathways, all of which increased susceptibility to apoptosis.8Desouza M. Gunning P.W. Stehn J.R. The actin cytoskeleton as a sensor and mediator of apoptosis.Bioarchitecture. 2012; 2: 75-87Crossref PubMed Google Scholar In addition, the conserved Switch 1 domain in the Rac guanosine triphosphatases binds to the p100β catalytic subunit and activates phosphoinositide-3-kinase (PI3K).9Fritsch R. de Krijger I. Fritsch K. George R. Reason B. Kumar M.S. et al.RAS and RHO families of GTPases directly regulate distinct phosphoinositide 3-kinase isoforms.Cell. 2013; 153: 1050-1063Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar PI3K overactivation, due to germline gain-of-function mutations in PIK3CD or PIK3R1, results in a combined immunodeficiency with certain features shared by our patients, including reduced percentages of naive T cells, the accumulation of senescent CD8+CD57+ lymphocytes, and increased T- and B-cell apoptosis.10Cannons J.L. Preite S. Kapnick S.M. Uzel G. Schwartzberg P.L. Genetic defects in phosphoinositide 3-kinase δ influence CD8+ T cell survival, differentiation, and function.Front Immunol. 2018; 9: 1758Google Scholar The lymphopenia in patients with PI3K overactivation has been attributed to TCR-mediated apoptotic signaling as well as metabolic shifts that favor the expansion of memory and senescent T cells over naive cells.10Cannons J.L. Preite S. Kapnick S.M. Uzel G. Schwartzberg P.L. Genetic defects in phosphoinositide 3-kinase δ influence CD8+ T cell survival, differentiation, and function.Front Immunol. 2018; 9: 1758Google Scholar However, because patients with activated phosphoinositide 3-kinase δ syndrome have additional features, including increased IgM levels, lymphoproliferative disease, and autoimmunity, additional patients and studies are needed to investigate the PI3K-AKT axis in patients with gain-of-function mutations in RAC2. Patients 2 and 3 also had features not seen in patient 1, such as reduced naive B cells and increased transitional B cells. Some of the variability in the immune phenotypes among the 3 patients may result from the exposure of patient 1 to chemotherapy, the age differences among the patients, or intrafamilial genetic variations. The data presented here provide a counterpoint to previous studies of patients with the dominant negative RAC2D57N mutant. Although impaired neutrophil function and chemotaxis are the hallmarks of loss-of-function variants in RAC2, we show that patients with a monoallelic activating RAC2 mutation have increased actin polymerization, exaggerated neutrophilic oxidative burst, lymphopenia, and increased lymphocyte apoptosis. This combined immunodeficiency indicates the importance of regulating RAC2 activity for intact immune function. We are grateful to the patients, the patients' families, and the nurses for their participation in this study. We thank David Williams and members of his laboratory for helpful discussions. Antibodies used for flow cytometry studies of T and B cells were as follows: anti-CD45RA FITC, anti-IgD FITC, anti-CD56 PE, anti-CD31 PE, anti-CD38 PerCP-Cy5.5, anti-CD3 PE-Cy7, anti-CD27 PE-Cy7, anti-CD4 APC, anti-CD8 APC-H7, anti-IgM Bv421, anti-CCR7 Bv421, anti-CD45 V500 Bv510, and anti-CD19 Bv510, all manufactured by Becton Dickinson (Franklin Lakes, NJ). Immunophenotyping of natural killer (NK) cells was done using the following antibodies: BAB281 (IgG1, anti-NKp46); AZ20 (IgG1, anti-NKp30); and anti-CD3 FITC and anti-CD56 PC5, anti-CD14 FITC, and anti-CD20 FITC (Beckman Coulter, Immunotech, Marseille, France). NK-cell analysis was performed by gating on CD56+CD3–CD14–CD20– cells. For assessment of CD11b and CD62L expression on neutrophils, whole blood samples from patients and controls were stained either with or without activation with phorbol 12-myristate 13-acetate (300 ng/mL) for 30 minutes at 37°C using anti-CD45 APC, anti-CD11b FITC, and anti-CD62L PerCP (all BD Pharmingen, San Diego, Calif). Analyses were performed using the FlowJo software version 8.8.7 (TreeStar, Ashland, Ore). T-cell proliferation was quantified using dilution of carboxyfluorsecein diacetate succinimidyl ester (Thermo Fisher Scientific, Waltham, Mass) after stimulation with anti-CD3 (10 μg/μL) (Sigma-Aldrich, St Louis, Mo) or PHA (5 ng/μL) (Sigma-Aldrich) as previously described.E1Lanzi G. Moratto D. Vairo D. Masneri S. Delmonte O. Paganini T. et al.A novel primary human immunodeficiency due to deficiency in the WASP-interacting protein WIP.J Exp Med. 2012; 209: 29-34Crossref PubMed Scopus (128) Google Scholar Whole-exome sequencing was performed on genomic DNA from all 3 patients using the Illumina HiSeq-2000 (Illumina Inc, San Diego, Calif), using Agilent SureSelect for library preparation. The average coverage of the exome was 173×. The Burrows-Wheeler Aligner was used to map reads to the human reference genome assembly GRCh37.E2Li H. Durbin R. Fast and accurate short read alignment with Burrows–Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (26860) Google Scholar Variants were called using the Genome Analysis Toolkit.E3DePristo M.A. Banks E. Poplin R. Garimella K.V. Maguire J.R. Hartl C. et al.A framework for variation discovery and genotyping using next-generation DNA sequencing data.Nat Genet. 2011; 43: 491-498Crossref PubMed Scopus (7140) Google Scholar Sanger sequencing of the P34H mutation in Exon 2 of RAC2 was performed with the following primers: (F: 5′-CTTGCAGGCTCTCGGGTGTG-3′, R: 5′-GTGCTGCCTTCTTGGCTGGA -3') with ABI PRISM 310 (Thermo Fisher Scientific). Lysates from T-cell blasts, BLCL, or transfected HEK293T cells were lysed in RIPA buffer (Millipore, Burlington, Mass), 50 mM EDTA (Thermo Fisher Scientific), and protease inhibitors (Thermo Fisher Scientific). Electrophoresis of lysates was performed on 4% to 12% precast polyacrylamide gels (Thermo Fisher Scientific), followed by immunoblotting with the following antibodies as indicated in figure legends: anti-Rac2 (AT2G1, Abcam, Cambridge, United Kingdom), anti-FLAG (F3165, Sigma-Aldrich), anti–β-actin (Sigma-Aldrich). HEK293T cells were transfected with either N-terminal FLAG-tagged WT Rac2 (Genecopoeia) or the Rac2P34H mutant, which was generated using the QuikChange II XL Site-Directed Mutagenesis kit (Agilent, Santa Clara, Calif). Two days after transfection using the TransIT-LT1 transfection reagent (Mirus Bio, Madison, Wis), cells were lysed in 25 mM HEPES, 150 mM NaCl, 1% IGEPAL CA-630, 10 mM MgCl2, 1 mM EDTA, 2% glycerol, and EDTA-free Protease Inhibitor Cocktail (Roche, Basel, Switzerland). To prepare guanosine triphosphate γS– or guanosine diphosphate–loaded controls, 10 mM EDTA was added to lysates, followed by 0.1 mM of guanosine triphosphate γS (Abcam) or 1 mM of guanosine diphosphate (Sigma-Aldrich) for 15 minutes at 37°C. Loading was stopped by the addition of 65 mM MgCl2. Active RAC2 was pulled down from a portion of the lysates by incubating with PAK-PBD beads (Cytoskeleton), followed by washing and immunoblotting using capillary Western blotting (ProteinSimple) and anti-FLAG antibody (Sigma-Aldrich). Whole blood from patients and controls was stimulated with N-formylmethionyl-leucyl-phenylalanine (1 μM) or phorbol 12-myristate 13-acetate (300 ng/mL), followed by Dihydrorhodamine 123 staining as previously described.E4Chen Y. Junger W.G. Measurement of oxidative burst in neutrophils.Methods Mol Biol Clifton NJ. 2012; 844: 115-124Crossref PubMed Scopus (96) Google Scholar Whole blood purified neutrophils from patients and controls were left untreated or stimulated with N-formylmethionyl-leucyl-phenylalanine (1 μM) for indicated times, followed by fixation and staining with fluorescein isothiocyanate–conjugated phalloidin (Sigma-Aldrich) per the manufacturer's guidelines. Apoptosis was determined in peripheral T cells from controls or patients, with or without anti-CD3 stimulation (10 μg/μL) (Sigma-Aldrich) for 48 hours. Apoptosis was assessed in EBV-immortalized B cells from controls or patients without stimulation. Live cells were identified by the absence of staining for either Annexin V or propidium iodide. (eBioscience, San Diego, Calif). For all graphs, each point in the scatterplots depicted represents a biologic replicate: each control point corresponds to a different healthy individual and each patient point corresponds to a different patient. Student t test was used for comparisons of 2 groups; 1- or 2-way ANOVA with the Holm-Šídák postcomparison test was used for the comparison of more than 2 groups, as indicated. Data are graphed as mean with SEM. Statistical analysis was performed using GraphPad Prism.Table E1Variants shared by all 3 patients, which are rare ( 15)ChromosomePositionReferencePatientConsequenceGene nameCADD score13328670ACNon-synonymousPRDM1619.7122310701CANon-synonymousCELA3B19.63127023871GTNon-synonymousARID1A21.1127679943CTNon-synonymousSYTL134127695833CTNon-synonymousFCN323.3127695833CTUpstreamMAP3K623.32220420994GANon-synonymousOBSL1323100373932GANon-synonymousGPR12818.75540681232GTNon-synonymousPTGER433579354543GANon-synonymousTHBS423.95102887994AGNon-synonymousNUDT1224.25112884695GANon-synonymousYTHDC222.95137713429CANon-synonymousKDM3B17.275147796767GANon-synonymousFBXO3827.85150227995TANon-synonymousIRGM22.791052014GTNon-synonymousDMRT2339121976315TANon-synonymousBRINP116.839130826079CGUpstreamSLC25A2522.19130826079CGNon-synonymousNAIF122.19140057403GCNon-synonymousGRIN1241014870199AGNon-synonymousCDNF24.31071966072GANon-synonymousPPA122.91210962022CTNon-synonymousTAS2R915.521210962022CTUpstreamTAS2R815.521229786258TCNon-synonymousTMTC129.31256030724TGNon-synonymousOR10P122.11257578143CTNon-synonymousLRP122.41421780617AGNon-synonymousRPGRIP127.31447600951ATNon-synonymousMDGA222.51472196852CGNon-synonymousSIPA1L122.61726684394TGUpstreamTMEM19915.59191592525ACNon-synonymousMBD328.3191592543GCNon-synonymousMBD323.3196452423CTNon-synonymousSLC25A2319.641944662108AGNon-synonymousZNF234251950832152TCUpstreamNR1H216.251950832152TCNon-synonymousKCNC316.252224953707CTUpstreamGUCD122.82224953707CTNon-synonymousSNRPD322.82237637633GTNon-synonymousRAC232CADD, Combined Annotation Dependent Depletion.The RAC2 variant is listed in boldface. Open table in a new tab CADD, Combined Annotation Dependent Depletion. The RAC2 variant is listed in boldface.