Dear Editor,

In the Rhesus (Rh) blood group system, D variant antigens are mainly due to a single-nucleotide variant or a RHD/RHCE hybrid gene and are classified into three groups: molecularly defined weak D phenotypes, molecularly defined partial D phenotypes, and DEL phenotypes [1]. Individuals having D variant antigens (except for Asian-type DEL and weak D types 1, 2, and 3) may have anti-D antibodies, which have clinical significance in transfusion or pregnancy [2]. Partial D may show variable serological reactivity. In some cases with positive reactivity rather than weak positive reactivity on RhD typing, it is difficult to detect partial D unless the individual has been exposed to D antigens, resulting in anti-D formation [3, 4]. Among various partial D alleles, RHD*DNT (RHD*38) shows a single missense variant in exon 3, namely c.455A>C (N152T) [5].

We report the first Korean case of RHD*DNT, which is the fourth case worldwide. This study was exempted from approval by the Institutional Review Board of the Konkuk University Medical Center (KUMC; approval number: 2022-09-051), Seoul, Korea, and the need for obtaining informed consent was waived. A 49-year-old Korean woman was admitted to the KUMC with adenomyosis. She had had two pregnancies (without Rh immunoglobulin [RhIG] administration) and a transfusion history (no information on the blood component). ABO and RhD typing using the Ortho BioVue Innova System (Ortho Clinical Diagnostics, Raritan, NJ, USA) revealed A and D 4+ reactivity, respectively. Antibody screening using the Ortho BioVue Innova System and antibody identification using 0.8% Resolve Panel A and Ficin-treated Panel C (Ortho Clinical Diagnostics, High Wycombe, UK) suggested the presence of anti-D. RhD typing using the tube method (Shinyang Diagnostics, Siheung, Korea) revealed 4+ reactivity. Rh phenotyping using an Ortho BioVue System RH/K II Cassette (Ortho Clinical Diagnostics) revealed the ccDEe phenotype.

Further evaluation was conducted in the Korean Rare Blood Program (KRBP) reference laboratory [6]. All anti-D reagents showed 4+ reactivity in both tube and column agglutination technology methods. Antibody screening using DiaCell I+II, Dia Positive (Bio-Rad, Glattbrugg, Switzerland) and antibody identification using ID-DiaPanel (Bio-Rad) with dithiothreitol-treated cells excluded anti-LW and confirmed anti-D. Real-time PCR and melting curve analysis revealed the presence of RHD, and PCR using sequence-specific primers (SSP) with BAGene RH-TYPE, Partial D-TYPE, and/or Weak D-TYPE SSP Kits (BAG Health Care GmbH, Lich, Germany) revealed partial D, suggesting RHD*DIIIc (RHD*03.03) or other alleles harboring N512T [6, 7]. Sanger sequencing of all 10 RHD exons and flanking regions of RHD confirmed RHD*DNT (method protocol available from the KRBP reference laboratory on request) [2].

The four RHD*DNT cases are summarized in Table 1 [8^-10]. Case 1 was a pregnant German woman having anti-D without a history of RhIG administration; case 2 was an African American male donor with D+hr^B–; case 3 was a Caucasian man referred for RHD zygosity testing; and case 4 is the present one. All four cases showed 4+ reactivity on all anti-D reagents tested, which made it difficult to identify D variant antigens. The two female cases (cases 1 and 4) were incidentally identified because they showed anti-D after pregnancy without RhIG administration, which hinted at partial D. All four cases were confirmed as RHD*DNT by Sanger sequencing of the RHD exons.

Table 1 . Summary of the four RHD*DNT cases reported worldwide to date

Basic and serological findings				Case 1 [ref. 8]	Case 2 [ref. 8]	Case 3 [ref. 8]	Case 4 (present case)
Race				German	African American	Caucasian	Korean
Age/sex				NA/F	NA/M	NA/M	49 yr/F
Anti-D				Positive	Negative	Negative	Positive
Rh phenotype				CcDee	NT	NT	ccDEe
RhD typing method				CAT	CAT	CAT	Tube & CAT
Anti-D reagent information and reactivity
Clone	Manufacturer*	Isotype	epD†	Reactivity
BS221	Biotest	IgG	6.3	4+	NT	NT	NT
BS226	NA [7]	IgM	6.4	4+	NT	NT	NT
BS227	Biotest	IgG	2.2	4+	NT	NT	NT
BS228	Biotest	IgG	6.3	4+	NT	NT	NT
BS229	Biotest	IgG	5.4	4+	NT	NT	NT
BS231	Biotest	IgG	5.4	4+	NT	NT	NT
BS232	NA [7]	IgM	6.4	4+	NT	NT	NT
D175-2	Immucor	IgM	6.1	4+	NT	NT	4+
D415 1E4	Immucor	IgG	NA	NT	NT	NT	4+
ESD1	Quotient	IgG	4.1	4+	4+	4+	NT
H41	Biotest	IgG	3.1	4+	NT	NT	NT
HM10	Diagast	IgM	6.6	4+	NT	NT	NT
HM16	Diagast	IgG	6.4	4+	NT	NT	NT
LDM-1	DiaMed	IgM	6‡	4+	NT	NT	NT
LHM169/80	DiaMed, Quotient	IgG	6.3	4+	4+	4+	NT
LHM169/81	Quotient	IgG	1.1	4+	4+	4+	NT
LHM174/102	Quotient	IgG	1.2	4+	4+	4+	NT
LHM50/2B	Quotient	IgG	6.3	4+	4+	4+	NT
LHM57/17	DiaMed, Quotient	IgG	6.3	4+	4+	4+	NT
LHM59/19	DiaMed, Quotient	IgG	8.1	4+	4+	4+	NT
LHM70/45	DiaMed, Quotient	IgG	1.2	4+	4+	4+	NT
LHM76/55	DiaMed, Quotient	IgG	3.1	4+	4+	4+	NT
LHM76/58	Quotient	IgG	8.1	4+	4+	4+	NT
LHM76/59	Quotient	IgG	5.1	4+	4+	4+	NT
LHM77/64	DiaMed, Quotient	IgG	9.1	4+	4+	4+	NT
LOR17-6C7	NA [7]	IgG	4.1	4+	NT	NT	NT
MAD2	Ortho	IgM	6‡	NT	NT	NT	4+
MS26	Shinyang	IgG	9‡	NT	NT	NT	4+
P3X212 11 F1	Diagast	IgM	8.2	4+	NT	NT	NT
P3X212 23 B10	Diagast	IgM	9.1	4+	NT	NT	4+
P3X241	Diagast	IgG	5.4	4+	NT	NT	NT
P3X249	Diagast	IgG	2.1	4+	NT	NT	NT
P3X290	Diagast	IgG	3.1	4+	NT	NT	4+
P3X35	Diagast	IgG	5.4	4+	NT	NT	4+
P3X61	Diagast	IgM	6.1	4+	NT	NT	4+
Polyclonal	Ortho	IgG	NA	NT	NT	NT	4+
RUM-1	Immucor	IgM	6.1	4+	NT	NT	4+
TH28	Shinyang	IgM	6‡	NT	NT	NT	4+

*Manufacturer information for the anti-D reagents: Immucor (Immucor, Inc., Norcross, GA, USA); Diagast (Diagast Laboratories, Lille, France); Quotient (Quotient Biodiagnostics, Newtown, PA, USA); DiaMed (DiaMed GmbH, Cressier sur Morat, Switzerland); Biotest (Biotest AG, Dreieich, Germany); Shinyang (Shinyang Diagnostics, Siheung, Korea); Ortho (Ortho Clinical Diagnostics, Raritan, NJ, USA); ^†D antigen epitopes were divided into epD 1–16 [⁹]; if not available, they were divided into epD 1–9 [¹⁰].

Abbreviations: M, male; F, female; epD, D antigen epitope; NA, not available; NT, not tested; CAT, column agglutination technology.

The 20 RHD alleles harboring c.455A>C (N152T) are listed in Table 2 [3^-5, 8]. The designation RHD*DNT was derived from the asparagine (N) to threonine (T) amino acid substitution, and N152T has been suggested to cause a high D antigen density (>20,000 antigens per red blood cell) by facilitating the integration of the RhD protein into the red blood cell membrane. Alleles harboring N152T may remain undetected (typed as D-positive) and anti-D immunization may occur, resulting in transfusion incompatibility and complications during pregnancy [8]. Individuals with partial D and anti-D were mostly females, and pregnancy was the most frequent cause of immunization; thus, women of childbearing age and girls would benefit from measures to prevent further anti-D immunization [8]. Although further research is required to define the underlying mechanism and to investigate the relationship between the increased antigen density and epitope change, it is assumed that the internally located N152T substitution (in the transmembrane zone) may alter extracellular epitopes. Among the 20 alleles harboring N152T, 11 alleles have been associated with anti-D formation [3, 4]; RHD alleles harboring N152T do not necessarily imply anti-D formation, and conversely, any alleles harboring N152T may have the potential to form anti-D. In RhD-positive individuals with anti-D formation, Sanger sequencing of the RHD exons can help clinically differentiate the RHD alleles harboring N152T. RHD*DNT could not have been identified without Sanger sequencing of the RHD exons; this implies that there may have been missed or unidentified RHD*DNT cases worldwide.

Table 2 . All 20 RHD alleles harboring c.455A>C (N152T)

Allele	Phenotype	Anti-D formation
RHD*DIIIa (RHD*03.01)	DIIIa	Reported [3]
RHD*DIIIc (RHD*03.03)	DIIIc	Reported [3]
RHD*DIII.4 (RHD*03.04)	DIII type 4	Reported [3]
RHD*DII.04.02 (RHD*03.04.02)	Not tested	Not reported [5]
RHD*DIII.6 (RHD*03.06)	DIII type 6	Not reported [3]
RHD*DIII.07 (RHD*03.07)	DIII type 7	Not reported [5]
RHD*DIII.08 (RHD*03.08)	DIII type 8	Reported [3]
RHD*DIII.09 (RHD*03.09)	DIII type 9	Not reported [5]
RHD*DIVa (RHD*04.01)	DIVa	Reported [3]
RHD*04.01.02	DIVa-like (DIVa type 3)	Not reported [5]
RHD*DVI.3 (RHD*06.03.01)	DVI type 3	Reported [3]
RHD*DVI.03.02 (RHD*06.03.02)	DVI type 3.2	Reported [3]
RHD*DVI.4 (RHD*06.04)	DVI type 4	Reported [3]
RHD*DOL4 (RHD*12.04)	DOL4	Reported [4]
RHD*DFR5 (RHD*17.05)	DFR5	Reported [3]
RHD*DNT (RHD*38)	RHD*DNT	Reported [8, present case]
RHD*D-SPM (RHD*40)	D-SPM	Not reported [5]
RHD*DKK (RHD*45)	DKK	Not reported [5]
RHD*62	DNT(V270G)	Not reported [5]
RHD*03N.01	D–, C+	Not reported [5]

In conclusion, we reported the first Korean case of RHD*DNT, which is the fourth case worldwide. RHD*DNT showed D-positive reactivity on RhD typing, which made it difficult to identify rare D variant antigens. Our case underscores the importance of molecular characterization for the identification of rare D variant antigens.

None.

Lee GH and Shin DW analyzed the data and wrote the draft; Hur M and Park KU conceived and designed the study, analyzed the data, and finalized the draft; Kim H, So KA, and Hong YJ collected the samples, analyzed the data, and participated in manuscript drafting. All authors have read and approved the final manuscript.

None declared.

None declared.