Acquired RhD positivity loss mediated by promoter hypermethylation is associated with adverse outcome in Myelodysplastic Syndrome: a case report and literature review

Acquired RhD positivity loss mediated by promoter hypermethylation is associated with adverse outcome in Myelodysplastic Syndrome: a case report and literature review | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Case Report Acquired RhD positivity loss mediated by promoter hypermethylation is associated with adverse outcome in Myelodysplastic Syndrome: a case report and literature review Na Wang, Ming Gong, Li Gao, Ying Cai, Linfeng Chen, Guochang Sun, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8004178/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The RhD antigen is a critical immunogenic protein on red blood cells. Acquired loss of RhD expression in individuals with a previous positive phenotype is a rare event, most often reported in hematological malignancies. Case presentation A 58-year-old woman with intermediate-risk myelodysplastic syndrome (MDS) was admitted with fever. Routine blood typing revealed a discrepant RhD-negative result, contradicting her documented positive status from three months prior. This was confirmed by a reference serological method. Results Genetic analysis identified a heterozygous RHD genotype, comprising one allele with a complete deletion and the other carrying a weak D allele ( RHD*01W.33 ). Quantitative methylation analysis demonstrated significant hypermethylation (75.20%) of RHD promoter in the current sample, compared to both her prior sample (64.65%;) and healthy control (67.08%). Despite supportive care, the patient died, reflecting her disease's aggressive course. A review of the literature confirmed the rarity of this phenomenon and highlighted that epigenetic dysregulation is a plausible, yet under-investigated mechanism. Conclusions This report directly implicates RHD promotor hypermethylation in acquired RhD antigen loss in MDS. Such epigenetic regulation may mark disease progression and herald a poor prognosis, justifying evaluation for hypomethylating therapy upon its detection. Myelodysplastic syndrome RhD weak D33 methylation epigenetic regulation Figures Figure 1 Figure 2 Figure 3 Background The RhD antigen (RH1), a highly immunogenic protein critical to blood group systems, is encoded by the RHD gene located on chromosome 1 (p34-36)[1, 2]. Standard serological testing with monoclonal antibodies classified individuals as RhD-positive or RhD- negative based on the presence or absence of the antigen on red blood cells (RBCs)[3]. Beyond these canonical phenotypes, variant phenotypes-often arising from quantitative or qualitive defects in antigen expression-can lead to discrepant typing results[4, 5]. These are typically categorized as “weak D” and “partial D”[6]. The expression of the RhD antigen is influenced by multiple factors, including DNA mutations[5, 7] and alterations at the RNA level[8]. Notably, certain disease states, particularly hematological malignancies, have been associated with modulated RhD antigen density[9–12]. However, the underlying mechanisms, especially epigenetic regulation, remain underexplored. In this case report, we describe a patient with myelodysplastic syndrome (MDS) who acquired a loss of RhD positivity. Through integrated serological, genetic and epigenetic analyses, we demonstrate that this phenomenon was mediated by promoter hypermethylation of the RHD gene, revealing a novel mechanism linking epigenetic dysregulation to blood group antigen loss in malignant hematological disease. Case presentation A 58-year-old woman with a known history of intermediate-risk MDS and interstitial pneumonia was admitted to our institution in June 2023 for persistent fever unresponsive to broad-spectrum antibiotics. A pre-transfusion blood group workup revealed an unexpected serologic discrepancy: her current typing was B, RhD-negative, which conflicted with her documented RhD-positive status from just three months ago. This finding prompted a comprehensive investigation to solve the discrepancy and elucidate the underlying mechanism. Diagnostic assessment and molecular investigations Serology The RhD-negative result was confirmed using another microcolumn agglutination test with a different anti-D clone and the standard tube method, ruling out a reagent-specific issue. Genetic analysis RHD gene sequencing identified a heterozygous genotype, comprising a complete deletion of exons 1–10 on one allele and the RHD01W.33 variant on the other (Table 2 ). Familial analysis of patient's son verified the inheritance of the RHD01W.33 allele, establishing the patient's genotype as a weak D phenotype. Table 2 Genotype results and the copy number determination using PCR-SBT Sample RHD allele ISBT terminology Genotype annotation Nucleotide change Exon location Amino acid change Copy number of RHD allele Hybrid Rhesus box results Patient Weak D type 33 RHD*01W.33 Weak D c.520G > A E4 p.Val174Met 1 RHD+/RHD- Patient's son Weak D type 33 RHD*01W.33 Weak D c.520G > A E4 p.Val174Met 2 RHD+/RHD+ Note: ISBT, International Society of Blood Transfusion Methylation analysis Given temporal loss of RhD expression, we hypothesized an epigenetic mechanism. Quantitative analysis of RHD promoter methylation was performed on the patient’s archived bone marrow sample from March 2023 and a current peripheral blood sample from June 2023. A significant hypermethylation was observed in the June sample (75.20%; Fig. 1 B) compared to the March sample (64.65%; Fig. 1 A) and a healthy control (67.08%; Fig. 1 C). Past medical history and treatment timeline The patient presented with a two-year history of anemia and recent cessation of acid/vitamin B12. Previously she was diagnosed with intermediated-risk MDS (April 2022) and interstitial pneumonia (March 2022), she was maintained on nintedanib with periodic transfusions. During an admission (March 2023) for severe anemia, serological testing identified an anti-E alloantibody. Her blood group was confirmed as B Ccee with an RhD-positive variant phenotype, prompting the transfusion of 2 units of B RhD-negative/E-antigen-negative RBCs. Subsequent therapy at Shunyi District Hospital (April-May 2023) included 12 additional units of B RhD-negative/E-antigen-free RBCs and 4 doses of B RhD-positive platelets. In her last admission (June 2023), she received supportive care with antimicrobials, high-flow oxygen, and further transfusions (4 units B RhD-negative/E-antigen-free RBCs + 4 doses B RhD-positive platelets). The patient expired on June 23, 2023, following electrocardiographic asystole. Literature review A literature search was conducted in the PubMed database for eligible English-language publications, with the most recent search performed on November 1st, 2025. Keywords including “RhD antigen shift/conversion”, “RhD antigen loss”, and “RhD antigen changes” were used in combination. The reference lists of retrieved articles were also manually screen. The process of study selection is summarized in Fig. 2 . A total of five studies reporting loss of RhD antigen expression-from previous positive to serologically negative-were identified. The clinical characteristics and proposed mechanisms from these cases are summarized in Table 3 . Among them, four cases occurred in patients with hematological malignancies. The loss of antigen was attributed to a multi-faceted process involving chromosomal aberrations on chromosome 1, suppression of RHD transcription, diminished mRNA stability, and functional impairment of hematopoietic stem cells. The remaining case involved a healthy pregnant woman, in whom the apparent RhD antigen discrepancy resulted from methodological discrepancies in serological testing. Table 3 Cases of conversion of RhD antigen expression from positive to negative by literature search. No. Reference Year of publication Number of cases Age (years) Gender (Male/Female) Diagnosis Possible mechanisms 1 Cooper et al[22] 1979 1 57 Male Myeloid metaplasia Balanced translocation involving chromosome 1and 13 2 Mertens et al[12] 1997 1 70 Male Myelodysplastic syndrome 1) Down regulation of RHD gene expression during the MDS process 2) Defective transcription activity or altered mRNA stability 3) Increased dysregulation of hematopoiesis 3 Cherif-Zahar et al[10] 1998 1 69 Female Breast cancer and chronic myelocytic leukemia Single nucleotide deletion in the RHD gene coding region 4 Sandler et al[23] 2012 1 40 Female Healthy pregnancy Revision in laboratory procedures for RhD tying 5 Radhakrishnan et al[9] 2016 1 11 Male Acute myeloid leukemia Mechanisms related with leukemia relapse Collectively, these reports suggest that acquired loss of RhD antigen in the setting of hematologic disease may arise from processes operating at multiple tiers, affecting transcription, translation or post-translational regulation of the RHD gene. Discussion In the present case, the patient was initially identified as a weak D phenotype (specifically weak D type 33). As her MDS progressed, we observed a corresponding increase in promoter methylation of the RHD gene, accompanied by a gradual weaking of RhD antigen expression. A complete shift in serological RhD typing from positive to negative is an exceptionally rare event; to our knowledge, this represents the first reported case linking such a loss of RhD antigen to an epigenetic modification. DNA methylation of CpG-rich promoters is a well-documented epigenetic mechanism in MDS[13–16]. In contrast to largely unmethylated promoter-associated CpG islands in normal tissues, MDS is characterized by widespread aberrant hypermethylation[14, 17], which can lead to stable, clonally propagated gene silencing[18]. In our patient, the methylation level of the RHD promoter hypermethylation contributed to the attenuated RHD gene expression and the consequent reduction of RhD antigen on erythrocytes. A schematic diagram of hypothesized mechanism is provided in Fig. 3 . Weak D type 33, resulting from an amino acid substitution in the transmembrane domain of the RhD protein[3, 19, 20], is a quantitative variant that reduced antigen density without altering the qualitative antigenic properties of the D epitope[19, 21]. This variant accounts for approximately 0.395% of D variants in the Chinese population[21]. Serologically, weak D33 is often interpreted as a normal RhD-positive result by highly sensitive methods such as microcolumn gel cards, as was observed in our patient until March 2023 (Table 1 ). Given the risk of anti-D alloimmunization in such cases, confirmation of RhD status using less sensitive methods (e.g., manual tile or tube testing) is strongly recommended[20]. Table 1 The patient's laboratory indices Mar. 19 2022 Jun. 16 2022 Oct. 28 2022 Mar. 13 2023 Apr. 6 2023 May. 18 2023 Jun. 11 2023 Reference values* WBC (Í10 9 / L) 2.92 4.38 10.45 2.28 2.52 18.99 8.25 3.50–9.50 RBC (Í10 12 / L) 1.95 2.13 3.69 1.29 2.01 1.56 2.54 3.80–5.10 Hb (g/L) 64.00 81.00 128.00 48.00 65.00 47.00 76.00 115.00-150.00 Platelets (Í10 9 / L) 88.00 139.00 269.00 9.00 8.00 65.00 4.00 100.00-350.00 Anti-D (IgM) 4+ 4+ 4+ 4 + w 1+ 0 0 - Note: WBC, white blood cells; RBC, red blood cells; Hb, hemoglobin; w, weak agglutin; 0, no agglutin. *Reference values are affected by many variables, including the patient population and laboratory methods used. The ranges used at China-Japan Friendship Hospital are for adults who are not pregnant and have no medical condition that could affect the results. They are thus not appropriate for all patients. It should be noted that the bone marrow aspiration in March 2023 revealed hematopoietic stagnation with markedly deceased erythroid proliferation (data not shown), indicating impaired erythropoiesis. Furthermore, from March 2023 onward, the patient received exclusively RhD-negative RBCs transfusions. Therefore, the possibility that circulating RBCs were primarily of donor origin cannot be entirely excluded as a contributing factor to this serological finding. Conclusions Promoter hypermethylation of the RHD gene can silence antigen expression in MDS. This loss of RhD may serve as a potential biomarker for adverse prognosis, warranting immediate evaluation for hypomethylating therapy. Abbreviations MDS Myelodysplastic syndrome RBCs Red blood cells Declarations Ethics approval and consent to participate The ethics committee of the China-Japan Friendship Hospital approved this study. Informed consent was obtained from the patient and her son following the Declaration of Helsinki. All patient data were anonymized and de-identified before analysis. Consent for publication The patient and her son provided consent for publication of this case report. Availability of data and materials The datasets generated and/or analysed during the current study are available in the NCBI repository, https://www.ncbi.nlm.nih.gov/gdv/browser/genome/?id=GCF_000001405.40. Data will be made on request, from the corresponding author, Hongkai Lu or Yongtong Cao. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding None. Authors’ contributions Na Wang and Hongkai Lu conceptualized the research. Ming Gong, Li Gao and Ying Cai participated in experimental design and analysis of results. Linfeng Chen and Guochang Sun collected the data. Na Wang conducted serological and molecular experiments. Na Wang wrote the manuscript. Yongtong Cao reviewed and edited the manuscript. Acknowledgements Not applicable. References Le van Kim C, Mouro I, Cherif-Zahar B, Raynal V, Cherrier C, Cartron JP, Colin Y: Molecular cloning and primary structure of the human blood group RhD polypeptide . Proc Natl Acad Sci U S A 1992, 89 (22):10925-10929. Flegel WA: Molecular genetics and clinical applications for RH . Transfus Apher Sci 2011, 44 (1):81-91. Flegel WA: The genetics of the Rhesus blood group system . Blood Transfus 2007, 5 (2):50-57. Sandler SG, Chen LN, Flegel WA: Serological weak D phenotypes: a review and guidance for interpreting the RhD blood type using the RHD genotype . Br J Haematol 2017, 179 (1):10-19. Raud L, Le Tertre M, Vigneron L, Ka C, Richard G, Callebaut I, Chen JM, Ferec C, Le Gac G, Fichou Y: Missense RHD single nucleotide variants induce weakened D antigen expression by altering splicing and/or protein expression . Transfusion 2021, 61 (8):2468-2476. 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16:43:14","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":13937,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineBMCMedicalGenomicscaseFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8004178/v1/d6735215a0df2a76484c5707.png"},{"id":98072281,"identity":"80e6f95b-0332-4b17-bb9d-706838d8f254","added_by":"auto","created_at":"2025-12-12 13:12:31","extension":"xml","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":55252,"visible":true,"origin":"","legend":"","description":"","filename":"887bd0cdbe4845f9a3693edb4c86fb3e1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8004178/v1/05ff630afd88a6cb7e2db22a.xml"},{"id":98072276,"identity":"d0041e73-1027-4578-a5e2-183f6b91df80","added_by":"auto","created_at":"2025-12-12 13:12:31","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":63053,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8004178/v1/caec0d062d9170eaab935d20.html"},{"id":98428882,"identity":"1bd9c0de-4e6b-4f1b-833e-f41014ac9358","added_by":"auto","created_at":"2025-12-17 16:42:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":787127,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative mean methylation rate after pyrosequencing. Bisulfite-treated DNA samples from patient's bone marrow in March (A), patient's peripheral blood in June (B), and healthy control's peripheral blood (C) were quantitatively analyzed by pyrosequencing. Three analyzed CpG sites are highlighted in blue, and the percent methylation rate is provided for each site. The mean percentage was computed as the \u003cem\u003eRHD \u003c/em\u003emethylation rate for each sample.\u003c/p\u003e","description":"","filename":"BMCMedicalGenomicscaseFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8004178/v1/cf7de0a3fd8e1ce5fc53e8b6.png"},{"id":98429506,"identity":"9ae04858-ae89-40fb-b4bc-a0eff617e472","added_by":"auto","created_at":"2025-12-17 16:43:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":272963,"visible":true,"origin":"","legend":"\u003cp\u003eFlow chart of collected articles.\u003c/p\u003e","description":"","filename":"BMCMedicalGenomicscaseFig2flowchart.png","url":"https://assets-eu.researchsquare.com/files/rs-8004178/v1/89123c732a6bc6d64f4971eb.png"},{"id":98429533,"identity":"7b675fb5-7d74-4f33-98f3-cb9ffb3704e6","added_by":"auto","created_at":"2025-12-17 16:43:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":82028,"visible":true,"origin":"","legend":"\u003cp\u003eA schematic diagram about the hypermethylation decreases the expression of the RhD antigen. The top panel, the normal expression of RhD antigen; the bottom panel, the complete loss of RhD antigen due to higher level of methylation of \u003cem\u003eRHD \u003c/em\u003egene promotor.\u003c/p\u003e","description":"","filename":"BMCMedicalGenomicscaseFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8004178/v1/228128198bf1dec6daba5383.png"},{"id":107900190,"identity":"28733d7f-c336-491b-9d25-9d73a9a12602","added_by":"auto","created_at":"2026-04-27 11:27:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1435480,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8004178/v1/7972afaa-2ec6-43d4-a495-7d32fd60d18c.pdf"},{"id":98072264,"identity":"8546519f-b23a-4f03-adcd-8ba73e331267","added_by":"auto","created_at":"2025-12-12 13:12:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":47644,"visible":true,"origin":"","legend":"","description":"","filename":"CAREchecklistNaWangBMC1101.docx","url":"https://assets-eu.researchsquare.com/files/rs-8004178/v1/c1954c91ea8a7535d583a2db.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Acquired RhD positivity loss mediated by promoter hypermethylation is associated with adverse outcome in Myelodysplastic Syndrome: a case report and literature review","fulltext":[{"header":"Background","content":"\u003cp\u003eThe RhD antigen (RH1), a highly immunogenic protein critical to blood group systems, is encoded by the \u003cem\u003eRHD\u003c/em\u003e gene located on chromosome 1 (p34-36)[1, 2]. Standard serological testing with monoclonal antibodies classified individuals as RhD-positive or RhD- negative based on the presence or absence of the antigen on red blood cells (RBCs)[3]. Beyond these canonical phenotypes, variant phenotypes-often arising from quantitative or qualitive defects in antigen expression-can lead to discrepant typing results[4, 5]. These are typically categorized as \u0026ldquo;weak D\u0026rdquo; and \u0026ldquo;partial D\u0026rdquo;[6].\u003c/p\u003e\u003cp\u003eThe expression of the RhD antigen is influenced by multiple factors, including DNA mutations[5, 7] and alterations at the RNA level[8]. Notably, certain disease states, particularly hematological malignancies, have been associated with modulated RhD antigen density[9\u0026ndash;12]. However, the underlying mechanisms, especially epigenetic regulation, remain underexplored.\u003c/p\u003e\u003cp\u003eIn this case report, we describe a patient with myelodysplastic syndrome (MDS) who acquired a loss of RhD positivity. Through integrated serological, genetic and epigenetic analyses, we demonstrate that this phenomenon was mediated by promoter hypermethylation of the \u003cem\u003eRHD\u003c/em\u003e gene, revealing a novel mechanism linking epigenetic dysregulation to blood group antigen loss in malignant hematological disease.\u003c/p\u003e"},{"header":"Case presentation","content":"\u003cp\u003eA 58-year-old woman with a known history of intermediate-risk MDS and interstitial pneumonia was admitted to our institution in June 2023 for persistent fever unresponsive to broad-spectrum antibiotics. A pre-transfusion blood group workup revealed an unexpected serologic discrepancy: her current typing was B, RhD-negative, which conflicted with her documented RhD-positive status from just three months ago. This finding prompted a comprehensive investigation to solve the discrepancy and elucidate the underlying mechanism.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eDiagnostic assessment and molecular investigations\u003c/h2\u003e\u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\u003ch2\u003eSerology\u003c/h2\u003e\u003cp\u003eThe RhD-negative result was confirmed using another microcolumn agglutination test with a different anti-D clone and the standard tube method, ruling out a reagent-specific issue.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003eGenetic analysis\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eRHD\u003c/em\u003e gene sequencing identified a heterozygous genotype, comprising a complete deletion of exons 1\u0026ndash;10 on one allele and the \u003cem\u003eRHD01W.33\u003c/em\u003e variant on the other (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Familial analysis of patient's son verified the inheritance of the \u003cem\u003eRHD01W.33\u003c/em\u003e allele, establishing the patient's genotype as a weak D phenotype.\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 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGenotype results and the copy number determination using PCR-SBT\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\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\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eRHD\u003c/em\u003e allele\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eISBT terminology\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGenotype annotation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNucleotide change\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eExon location\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAmino acid change\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eCopy number of \u003cem\u003eRHD\u003c/em\u003e allele\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eHybrid Rhesus box results\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePatient\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWeak D type 33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eRHD*01W.33\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWeak D\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ec.520G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eE4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ep.Val174Met\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eRHD+/RHD-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePatient's son\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWeak D type 33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eRHD*01W.33\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWeak D\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ec.520G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eE4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ep.Val174Met\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eRHD+/RHD+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003eNote: ISBT, International Society of Blood Transfusion\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eMethylation analysis\u003c/h3\u003e\n\u003cp\u003eGiven temporal loss of RhD expression, we hypothesized an epigenetic mechanism. Quantitative analysis of \u003cem\u003eRHD\u003c/em\u003e promoter methylation was performed on the patient\u0026rsquo;s archived bone marrow sample from March 2023 and a current peripheral blood sample from June 2023. A significant hypermethylation was observed in the June sample (75.20%; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) compared to the March sample (64.65%; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and a healthy control (67.08%; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003ePast medical history and treatment timeline\u003c/h3\u003e\n\u003cp\u003eThe patient presented with a two-year history of anemia and recent cessation of acid/vitamin B12. Previously she was diagnosed with intermediated-risk MDS (April 2022) and interstitial pneumonia (March 2022), she was maintained on nintedanib with periodic transfusions. During an admission (March 2023) for severe anemia, serological testing identified an anti-E alloantibody. Her blood group was confirmed as B Ccee with an RhD-positive variant phenotype, prompting the transfusion of 2 units of B RhD-negative/E-antigen-negative RBCs. Subsequent therapy at Shunyi District Hospital (April-May 2023) included 12 additional units of B RhD-negative/E-antigen-free RBCs and 4 doses of B RhD-positive platelets.\u003c/p\u003e\u003cp\u003eIn her last admission (June 2023), she received supportive care with antimicrobials, high-flow oxygen, and further transfusions (4 units B RhD-negative/E-antigen-free RBCs\u0026thinsp;+\u0026thinsp;4 doses B RhD-positive platelets). The patient expired on June 23, 2023, following electrocardiographic asystole.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eLiterature review\u003c/h2\u003e\u003cp\u003eA literature search was conducted in the PubMed database for eligible English-language publications, with the most recent search performed on November 1st, 2025. Keywords including \u0026ldquo;RhD antigen shift/conversion\u0026rdquo;, \u0026ldquo;RhD antigen loss\u0026rdquo;, and \u0026ldquo;RhD antigen changes\u0026rdquo; were used in combination. The reference lists of retrieved articles were also manually screen. The process of study selection is summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA total of five studies reporting loss of RhD antigen expression-from previous positive to serologically negative-were identified. The clinical characteristics and proposed mechanisms from these cases are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Among them, four cases occurred in patients with hematological malignancies. The loss of antigen was attributed to a multi-faceted process involving chromosomal aberrations on chromosome 1, suppression of \u003cem\u003eRHD\u003c/em\u003e transcription, diminished mRNA stability, and functional impairment of hematopoietic stem cells. The remaining case involved a healthy pregnant woman, in whom the apparent RhD antigen discrepancy resulted from methodological discrepancies in serological testing.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCases of conversion of RhD antigen expression from positive to negative by literature search.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNo.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eYear of publication\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNumber of cases\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAge (years)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eGender\u003c/p\u003e\u003cp\u003e(Male/Female)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eDiagnosis\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003ePossible mechanisms\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCooper et al[22]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1979\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMyeloid metaplasia\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eBalanced translocation involving chromosome 1and 13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMertens et al[12]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1997\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMyelodysplastic syndrome\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1) Down regulation of \u003cem\u003eRHD\u003c/em\u003e gene expression during the MDS process\u003c/p\u003e\u003cp\u003e2) Defective transcription activity or altered mRNA stability\u003c/p\u003e\u003cp\u003e3) Increased dysregulation of hematopoiesis\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCherif-Zahar et al[10]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1998\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eBreast cancer and chronic myelocytic leukemia\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eSingle nucleotide deletion in the \u003cem\u003eRHD\u003c/em\u003e gene coding region\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSandler et al[23]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2012\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHealthy pregnancy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eRevision in laboratory procedures for RhD tying\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRadhakrishnan et al[9]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2016\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAcute myeloid leukemia\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eMechanisms related with leukemia relapse\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eCollectively, these reports suggest that acquired loss of RhD antigen in the setting of hematologic disease may arise from processes operating at multiple tiers, affecting transcription, translation or post-translational regulation of the \u003cem\u003eRHD\u003c/em\u003e gene.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present case, the patient was initially identified as a weak D phenotype (specifically weak D type 33). As her MDS progressed, we observed a corresponding increase in promoter methylation of the \u003cem\u003eRHD\u003c/em\u003e gene, accompanied by a gradual weaking of RhD antigen expression. A complete shift in serological RhD typing from positive to negative is an exceptionally rare event; to our knowledge, this represents the first reported case linking such a loss of RhD antigen to an epigenetic modification.\u003c/p\u003e\u003cp\u003eDNA methylation of CpG-rich promoters is a well-documented epigenetic mechanism in MDS[13\u0026ndash;16]. In contrast to largely unmethylated promoter-associated CpG islands in normal tissues, MDS is characterized by widespread aberrant hypermethylation[14, 17], which can lead to stable, clonally propagated gene silencing[18]. In our patient, the methylation level of the \u003cem\u003eRHD\u003c/em\u003e promoter hypermethylation contributed to the attenuated \u003cem\u003eRHD\u003c/em\u003e gene expression and the consequent reduction of RhD antigen on erythrocytes. A schematic diagram of hypothesized mechanism is provided in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWeak D type 33, resulting from an amino acid substitution in the transmembrane domain of the RhD protein[3, 19, 20], is a quantitative variant that reduced antigen density without altering the qualitative antigenic properties of the D epitope[19, 21]. This variant accounts for approximately 0.395% of D variants in the Chinese population[21]. Serologically, weak D33 is often interpreted as a normal RhD-positive result by highly sensitive methods such as microcolumn gel cards, as was observed in our patient until March 2023 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Given the risk of anti-D alloimmunization in such cases, confirmation of RhD status using less sensitive methods (e.g., manual tile or tube testing) is strongly recommended[20].\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe patient's laboratory indices\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\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\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMar. 19 2022\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJun. 16\u003c/p\u003e\u003cp\u003e2022\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOct. 28\u003c/p\u003e\u003cp\u003e2022\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMar. 13\u003c/p\u003e\u003cp\u003e2023\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eApr. 6 2023\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMay. 18 2023\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eJun. 11\u003c/p\u003e\u003cp\u003e2023\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eReference values*\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWBC (\u0026Iacute;10\u003csup\u003e9\u003c/sup\u003e/ L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e18.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e8.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e3.50\u0026ndash;9.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRBC (\u0026Iacute;10\u003csup\u003e12\u003c/sup\u003e/ L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e3.80\u0026ndash;5.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHb (g/L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e64.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e81.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e128.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e48.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e65.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e47.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e76.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e115.00-150.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePlatelets (\u0026Iacute;10\u003csup\u003e9\u003c/sup\u003e/ L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e88.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e139.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e269.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e65.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e100.00-350.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-D (IgM)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4\u0026thinsp;+\u0026thinsp;w\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003eNote: WBC, white blood cells; RBC, red blood cells; Hb, hemoglobin; w, weak agglutin; 0, no agglutin.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003e*Reference values are affected by many variables, including the patient population and laboratory methods used. The ranges used at China-Japan Friendship Hospital are for adults who are not pregnant and have no medical condition that could affect the results. They are thus not appropriate for all patients.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIt should be noted that the bone marrow aspiration in March 2023 revealed hematopoietic stagnation with markedly deceased erythroid proliferation (data not shown), indicating impaired erythropoiesis. Furthermore, from March 2023 onward, the patient received exclusively RhD-negative RBCs transfusions. Therefore, the possibility that circulating RBCs were primarily of donor origin cannot be entirely excluded as a contributing factor to this serological finding.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003ePromoter hypermethylation of the \u003cem\u003eRHD\u003c/em\u003e gene can silence antigen expression in MDS. This loss of RhD may serve as a potential biomarker for adverse prognosis, warranting immediate evaluation for hypomethylating therapy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eMDS Myelodysplastic syndrome\u003c/p\u003e\n\u003cp\u003eRBCs Red blood cells\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003eThe ethics committee of the China-Japan Friendship Hospital approved this study. Informed consent was obtained from the patient and her son following the Declaration of Helsinki. All patient data were anonymized and de-identified before analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003eThe patient and her son provided consent for publication of this case report.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003eThe datasets generated and/or analysed during the current study are available in the NCBI repository, https://www.ncbi.nlm.nih.gov/gdv/browser/genome/?id=GCF_000001405.40. Data will be made on request, from the corresponding author, Hongkai Lu or Yongtong Cao.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u0026nbsp;\u003c/strong\u003eNa Wang and Hongkai Lu conceptualized the research. Ming Gong, Li Gao and Ying Cai participated in experimental design and analysis of results. Linfeng Chen and Guochang Sun collected the data. Na Wang conducted serological and molecular experiments. Na Wang wrote the manuscript. Yongtong Cao reviewed and edited the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLe van Kim C, Mouro I, Cherif-Zahar B, Raynal V, Cherrier C, Cartron JP, Colin Y: \u003cstrong\u003eMolecular cloning and primary structure of the human blood group RhD polypeptide\u003c/strong\u003e. \u003cem\u003eProc Natl Acad Sci U S A \u003c/em\u003e1992, \u003cstrong\u003e89\u003c/strong\u003e(22):10925-10929.\u003c/li\u003e\n\u003cli\u003eFlegel WA: \u003cstrong\u003eMolecular genetics and clinical applications for RH\u003c/strong\u003e. \u003cem\u003eTransfus Apher Sci \u003c/em\u003e2011, \u003cstrong\u003e44\u003c/strong\u003e(1):81-91.\u003c/li\u003e\n\u003cli\u003eFlegel WA: \u003cstrong\u003eThe genetics of the Rhesus blood group system\u003c/strong\u003e. \u003cem\u003eBlood Transfus \u003c/em\u003e2007, 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Richard G, Fichou Y, Luo G, Ji Y: \u003cstrong\u003eMolecular and serological analysis of the D variant in the Chinese population and identification of seven novel RHD alleles\u003c/strong\u003e. \u003cem\u003eTransfusion \u003c/em\u003e2023, \u003cstrong\u003e63\u003c/strong\u003e(2):402-414.\u003c/li\u003e\n\u003cli\u003eCooper B, Tishler PV, Atkins L, Breg WR: \u003cstrong\u003eLoss of Rh antigen associated with acquired Rh antibodies and a chromosome translocation in a patient with myeloid metaplasia\u003c/strong\u003e. \u003cem\u003eBlood \u003c/em\u003e1979, \u003cstrong\u003e54\u003c/strong\u003e(3):642-647.\u003c/li\u003e\n\u003cli\u003eSandler SG, Li W, Langeberg A, Landy HJ: \u003cstrong\u003eNew laboratory procedures and Rh blood type changes in a pregnant woman\u003c/strong\u003e. \u003cem\u003eObstet Gynecol \u003c/em\u003e2012, \u003cstrong\u003e119\u003c/strong\u003e(2 Pt 2):426-428.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Myelodysplastic syndrome, RhD, weak D33, methylation, epigenetic regulation","lastPublishedDoi":"10.21203/rs.3.rs-8004178/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8004178/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe RhD antigen is a critical immunogenic protein on red blood cells. Acquired loss of RhD expression in individuals with a previous positive phenotype is a rare event, most often reported in hematological malignancies.\u003c/p\u003e\u003ch2\u003eCase presentation\u003c/h2\u003e\u003cp\u003eA 58-year-old woman with intermediate-risk myelodysplastic syndrome (MDS) was admitted with fever. Routine blood typing revealed a discrepant RhD-negative result, contradicting her documented positive status from three months prior. This was confirmed by a reference serological method.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eGenetic analysis identified a heterozygous \u003cem\u003eRHD\u003c/em\u003e genotype, comprising one allele with a complete deletion and the other carrying a weak D allele (\u003cem\u003eRHD*01W.33\u003c/em\u003e). Quantitative methylation analysis demonstrated significant hypermethylation (75.20%) of \u003cem\u003eRHD\u003c/em\u003e promoter in the current sample, compared to both her prior sample (64.65%;) and healthy control (67.08%). Despite supportive care, the patient died, reflecting her disease's aggressive course. A review of the literature confirmed the rarity of this phenomenon and highlighted that epigenetic dysregulation is a plausible, yet under-investigated mechanism.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThis report directly implicates \u003cem\u003eRHD\u003c/em\u003e promotor hypermethylation in acquired RhD antigen loss in MDS. Such epigenetic regulation may mark disease progression and herald a poor prognosis, justifying evaluation for hypomethylating therapy upon its detection.\u003c/p\u003e","manuscriptTitle":"Acquired RhD positivity loss mediated by promoter hypermethylation is associated with adverse outcome in Myelodysplastic Syndrome: a case report and literature review","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-12 13:12:26","doi":"10.21203/rs.3.rs-8004178/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"60a72a50-7892-4afd-997c-b9fe0919c9bf","owner":[],"postedDate":"December 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T11:27:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-12 13:12:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8004178","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8004178","identity":"rs-8004178","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}