SCAR: The high-prevalence antigen 013.008 in the Scianna blood group system

Abstract

Background:

The Scianna (SC) blood group system comprises seven antigens. They reside on the erythroblast membrane-associated glycoprotein (ERMAP). The ERMAP and RHCE genes are juxtaposed to each other on chromosome 1. We report a novel SC antigen.

Study Design and Methods:

Blood samples came from a patient and his two sisters in Saudi Arabia. To investigate the antibody specificity we used the column agglutination technique and soluble recombinant ERMAP protein. The significance of anti-SCAR was evaluated by the transfusion history and a monocyte monolayer assay. We determined the genomic sequence of ERMAP and RHCE genes.

Results:

The patientʼs serum showed an antibody of titer 8 against a high-prevalence antigen. The soluble recombinant ERMAP protein inhibited the antibody. The propositus genotyped homozygous for an ERMAP:c.424C>G variant, for which his sisters were heterozygous. The c.424C>G variant occurred in the SC*01 allele in one haplotype with the RHCE*03 (RHCE*cE) allele. No signs of hemolysis occurred following an incompatible blood transfusion. The monocyte monolayer assay was negative.

Conclusions:

We characterized a high-prevalence antigen, with the proposed name “SCAR,” which is the eighth antigen of the Scianna blood group system (proposed designation 013.008). Individuals homozygous for ERMAP:p. (Gln142Glu) protein variant can produce anti-SCAR. Although we did not observe any sign of hemolysis at this time, the anti-SCAR prompted a change of the treatment regimen. A review of the known reports indicated that all SC alloantibodies of sufficient titer should be considered capable of causing hemolysis.

1 |. INTRODUCTION

The human ERMAP gene is located on the short arm of chromosome 1 (1p34.2), 42.8 Mbp from the telomeric end and 79.2 Mbp from the centromeric end.¹ It is positioned 17.5 Mbp centromeric to the RHCE gene.¹ The ERMAP gene encodes a single pass transmembrane glycoprotein, which carries the seven known antigens of the Scianna blood group system (SC; ISBT 013).^2–4 ERMAP is a glycosylated protein highly expressed on erythroid tissues and weakly expressed on leukocytes, thymus, lymph nodes, spleen, and bone marrow cells of adults and in fetal liver.⁵ It functions as a cell adhesion or receptor molecule and potentially as an immune regulator.⁶

We recently reviewed reports on allo- and autoantibodies to SC antigens.^3,7 While antibodies against SC antigens are infrequently found, some are clinically significant.^3,8–14 They can be missed easily, particularly when other antibodies are present.¹⁵ SC antibodies to high-prevalence antigens, particularly Sc1, and also Sc3, Sc5, Sc6, and Sc7, can mask other antibodies, when inhibition using soluble protein is possible and helpful.^15,16 Previous reports suggest alloantibodies to Sc2¹⁷ and Sc4¹⁸ are associated with severe hemolytic disease of the fetus and newborn (HDFN) and some with acute hemolytic^19,20 or delayed hemolytic or serologic transfusion reactions.^3,7,13,21 Autoantibodies binding to the SC protein have been reported in patients with clinically relevant autoimmune hemolytic anemia.^7,19,22–25

We report a patient with an antibody to a novel high-prevalence antigen in the SC blood group system. Our workup included molecular characterization of a single nucleotide variant (SNV) in the underlying rare ERMAP allele linked to a distinct R² allele of the RHCE gene, and antibody inhibition by soluble ERMAP protein.

2 |. CASE REPORT

The patient was born in 1996 to parents who were first-degree cousins as the youngest of four siblings: two sisters had sickle cell trait and one brother was healthy. The family had come from the southern region of Saudi Arabia, where sickle cell disease is more prevalent (24 cases/10000 population),²⁶ and lived in the central region (Riyadh). The patient was diagnosed with sickle cell beta thalassemia at age 5 months. His red cell phenotype was A, RhD-positive, C−e−E+c+, K−. Disease manifestations until age 23 years included acute chest syndrome, recurrent multifocal osteomyelitis, septic arthritis, left femoral head avascular necrosis, and bilateral mild hydronephrosis.

2.1 |. Transfusion history

Beginning from age 1 year, the patient had undergone blood and platelet transfusion on multiple occasions. The first antibodies identified were anti-C and anti-e in 1998. In 2010, a pan-reactivity was observed and reported as “antibody of undetermined specificity.”. In May 2013, the patient had a negative antibody screen. At a hemoglobin concentration of 8.4 g/dL, he was transfused with a crossmatch compatible, phenotype matched red cell unit. The hemoglobin increased to 9.6 g/dL but fell to 8.6 g/dL on the following day.

The patient was admitted in November 2013 for a hemoglobin of 6.3 g/dL. A 3+ pan-reactivity was observed in anti-globulin phase, while the direct antiglobulin test (DAT) and auto-control were negative, ruling out the possibility of a warm autoantibody. All crossmatched red cell units were incompatible. A crossmatch incompatible, phenotype matched (C−e−E+c+, K−) red cell unit was transfused. The hemoglobin increased to 7.4 g/dL and remained for 1 month. The auto-control turned to 3+, as was a polyspecific and an anti-IgG monospecific DAT. An eluate reacted with all red cells tested.

Because serologically compatible red cell units were lacking, the patient received hydroxyurea since 2015, which improved his clinical outcome with an average of only 1–2 painful crises per year. The HbF, which used to be 3.2% in 2006, was 19.5% in 2020.

2.2 |. Immunohematology

The serum showed pan-reactivity on reverse blood grouping in tube polyethylene glycol (PEG)-IAT with an 11 red cell in-house panel (Versiti, Milwaukee, WI). The antibody remained reactive when tested with a 3-cell ficin-treated and 2-aminoethylisothiouronium bromide (AET)-treated red cells. The patient phenotyped by serology as SC:1,−2 and by red cell genotyping as SC:1,−2 (PreciseType HEA; Immucor, Norcross, GA).

An anti-Fy^a of titer 16 was identified in the plasma. Another antibody of titer 8 was recognized, possibly directed against an unknown high-prevalence antigen. Red cells matched for the patientʼs genotype/phenotype of the Rh, MNS, Kell, Duffy, Kidd, Lutheran, and Dombrock systems were used for allo-adsorption studies.

3 |. MATERIALS AND METHODS

3.1 |. Study subjects

EDTA-anticoagulated whole blood samples were collected in Saudi Arabia from the patient and his two sisters with written informed consent. The DNA was extracted using a BioRobot EZ1 workstation with EZ1 DNA blood kit (Qiagen, Valencia, CA).

3.2 |. Immunohematology

Antibody screening and identification was done using saline and PEG indirect antiglobulin test (IAT) and with 2-aminoeythylisthiouronium (AET) bromide hydrobromide-treated reagent red cells, and gel matrix tests (rabbit anti-IgG, Micro Typing Systems; Ortho Clinical Diagnostics, Pompano Beach, FL). Antigens were tested by standard tube or gel matrix tests with licensed reagents (Ortho, Raritan, NJ), if available.

We applied an adsorption/elution method (Gamma ELU-KIT II; Immucor) using 100 μL of packed red cells and 100 μL of anti-Sc3, as described previously.²⁷ We washed the sensitized red cells, eluted the bound immunoglobulin and tested the eluate against a 3-cell panel using indirect antiglobulin test (IAT) in IgG gel matrix technique.²⁷

3.3 |. Agglutination inhibition test

We added 1 μL soluble recombinant ERMAP protein (1 μg/μL; Novoprotein, NJ, USA) to 25 μL patientʼs plasma and incubated for 30 minutes at room temperature. Then, 50 μL of a 0.8% RBC suspension (MTS Diluent 2; Micro Typing Systems) was added, incubated for 15 minutes at 37°C and tested for agglutination in a gel matrix test. A dilution effect was excluded by testing the plasma with 1 μL of buffer instead of protein solution.

Agglutination strengths were noted ranging from “w” indicating weak reactivity to 4+ indicating very strong reactivity.²⁸ Agglutination strength 0 indicated a negative indirect antiglobulin test (IAT) and reflected complete inhibition or lack of reactivity.

3.4 |. Complement activation

The ability of anti-SCAR to activate complement was evaluated as previously described.²⁹ Following incubation of 50 μL of a 5% suspension of SC:8 red cells with 200 μL of patientʼs serum in a saline IAT for 30 minutes at 37°C, the anti-SCAR sensitized red cells were washed and resuspended in 200 μL of fresh AB serum for 30 minutes at 37°C.²⁹ We tested for IgG (Gamma-clone anti-IgG; murine monoclonal IgM antibody, clone 16H8; Immucor), C3b/d (Gamma-clone anti-C3b,-C3d; murine monoclonal antibodies, clones GAMA003/055A.305 and GAMA004/053A.714; Immucor), and C3d bound to red cells (Bioclone anti-C3d; murine monoclonal antibody, clone undisclosed; Ortho).

3.5 |. Monocyte monolayer assay (MMA)

Allogeneic peripheral blood mononuclear cells were incubated in chamber slides for 1 hour at 37°C, as previously described.^20,30 Red cells sensitized in vitro with patientʼs plasma were layered onto the monocyte monolayer and incubated for 1 hour at 37°C. The slides were rinsed to remove non-adherent red cells and the monocytes were stained. We counted 300 monocytes and calculated an MMA phagocytic index, indicating the number of red cells phagocytosed or attached per 100 monocytes. An MMA phagocytic index of >5 was considered indicative for clinical significance of an antibody.³¹

3.6 |. IgG subclass

The anti-SCAR IgG subclass was determined using sensitized RBCs in a saline tube method. The RBCs showed 3 + agglutination when tested with rabbit polyclonal anti-human IgG (Ortho Clinical Diagnostics, Raritan, NJ). Subclass murine antibodies (Sigma Aldrich, St. Louis, MO: anti-IgG1 clone 8c/6–39, anti-IgG2 clone HP6002, anti-IgG3 clone HP-6050, anti-IgG4 clone HP-6025) diluted 1:125 in 6% bovine serum albumin in phosphate buffered saline were used.

3.7 |. Molecular screening of blood group genes

We determined SNVs, characteristic for the prediction of 44 antigens among 12 blood group systems, using real-time polymerase chain reaction (PCR)-fluorogenic 5′ nuclease (TaqMan) chemistry in a nanofluidic open array format^32,33 and (PreciseType HEA [human erythrocyte antigen] kit; Immucor).

3.8 |. Nucleotide sequencing of ERMAP and RHCE

Nucleotide sequencing of the full-length ERMAP gene covering exon 2 to 12 and the intervening sequence (IVS) was performed as described previously.² For RHCE, nucleotide sequences of all 10 exons as well as the adjacent intronic regions including the 5′- and 3′⁻ untranslated regions (UTR) were determined as described previously.³⁴ Nucleotide sequences were aligned (CodonCode Aligner; CodonCode, Dedham, MA) to NCBI RefSeq NG_008749.1 (ERMAP) or NG_009208.3 (RHCE) and nucleotide positions defined using the first nucleotide of the CDS of NM_001017922.1 (ERMAP) or NM_020485.4 (RHCE).

3.9 |. Molecular screening of RHD

Nucleotide sequence of RHD exon 7 and its adjacent intronic regions were determined as described previously.³⁴ Nucleotide sequences were aligned (CodonCode Aligner) to NCBI RefSeq NG_007494.1 and nucleotide positions defined using the first nucleotide of the CDS of NM_016124.4.

3.10 |. RHD zygosity

Zygosity testing for the RHD gene was done by restriction fragment length polymorphism (RFLP).³⁵

3.11 |. Prediction of functional impact of variants

Franklin by Genoox (https://franklin.genoox.com/home),³⁶ an artificial intelligence-based variant classification and interpretation based on guidelines by the American College of Medical Genetics and Genomics (ACMG),³⁷ was used to predict the functional impact of sequence variants. PredictSNP was also applied to predict the functional impact of non-synonymous nucleotide substitutions.³⁸

3.12 |. Modeling

The 3D structure for the partial ERMAP protein was predicted by online service of the Swiss Institute of Bioinformatics (http://swissmodel.expasy.org)³⁹ using the sodium channel subunit beta-1 6j8g.1.C as template and analyzed with Swiss-PDB-viewer.⁴⁰

3.13 |. Nomenclature

We proposed the name “SCAR” for the new antigen, derived from the SC blood group system and the second and third letters of the patient’s last name. At press deadline, the antigen numbering 013.008 and naming are pending approval by the ISBT Working Party on Red Cell Immunogenetics and Blood Group Terminology.⁴ Both items should be considered temporary until the final nomenclature is assigned by the Working Party.

4 |. RESULTS

We report ERMAP alleles in three individuals linked with distinct RHCE alleles. A 23-year-old Saudi Arabian patient with anti-Fy^a and a high-prevalence antibody carried a novel homozygous variation in an ERMAP allele. His Rh phenotype was C−, E+, c+, e−, V−, VS−, consistent with a history of anti-C and anti-e alloimmunization.

4.1 |. ERMAP alleles

The patient was homozygous for a variant ERMAP allele (MK933825; Table 1 and Table S1) with a SNV encoding a Gln to Glu amino acid substitution. It occurred in the SC*01 allele in 1 haplotype with the RHCE*03 (RHCE*cE) allele (in cis, on 1 chromosome). Both sisters were heterozygous for this variant and the reference ERMAP alleles (MK933825 and MN013142; Table 2 and Table S1).

TABLE 1.

ERMAP alleles identified in this study

	Nucleotide substitutions in ERMAP genea
	Coding sequence				Non-coding sequence
GenBank accession number	n	Exon involved	Position	Effect on protein	nb	HGVS nomenclaturec
MK933825	1	4	c.424C>G	p.Gln142Glu	15	NG_008749.1(ERMAP_v001):c.424C>G
MN013142	0	NA	NA	NA	21	NG_008749.1(ERMAP_v001):c.=

Abbreviation: NA, not applicable.

^aRelative to NCBI Reference Sequence NG_008749.1.

^bFor detailed information, see Table S1.

^cAccording to HGVS nomenclature recommendations.⁴¹

TABLE 2.

ERMAP alleles in the patient and his two sisters

		ERMAP
Subjects	Ethnicity	Zygosity	Alleles	Scianna phenotype	RH haplotype	Observations (n)
Patient	Saudi Arabian	Homozygous	MK933825 +MK933825	SC:1,3,−8a	DcE	1
Sisters	Saudi Arabian	Heterozygous	MK933825 +MN013142	SC:1,3,−4,8	DcE	2

^aRadin (Sc4) testing not done in the patient, because the test serum was ABO incompatible.

4.2 |. Agglutination inhibition test

Based on the molecular results, an antibody inhibition assay was performed using soluble recombinant ERMAP protein.¹⁵ The ERMAP protein specifically inhibited the high-prevalence antibody and a Fy^a specific reaction pattern was observed in a panel of test cells (data not shown). This result confirmed the high-prevalence antibody to be directed against the ERMAP protein. It represented a new antibody specificity, defining an SC antigen, with the proposed name SCAR and proposed antigen number SC8 (ISBT 013.008).

4.3 |. Serologic characterization

We tested the strength of SC antigens expressed on the patientʼs red cells. Anti-Sc1, anti-Sc3 and anti-SCAR titers did not differ much among the red cells of the patient, siblings, and controls (Table 3); there may be a dose effect for anti-SCAR. The anti-SCAR did not react with the patientʼs own red cells (Table 3). The Sc3 antigen was resistant to papain and DTT, and the titer did not vary among the patient and control red cells (Table 3); it varied in sensitivity to trypsin treatment (Table 3).⁴²

TABLE 3.

Serologic reactivity of anti-Sc antibodies

	Red cell phenotype		Antibody titer (number of individuals, if >1)
				Anti-Sc3				Anti-SCAR
Red cells	By sequencing	By serologya	Anti-Scl	Untreated	Papain treated	DTT treated	Trypsin treated	Untreated	Papain treated	DTT treated	Trypsin treated
Patient	SC:l,−2,3,−4,5,6,7,−8	SC:1,3	8	128	64	64	8	-	-	-	-
Sister 1b	SC:1,−2,3,−4,5,6,7,8	SC:1,3,−4	4	32	-	-	-	1	2	0	0
Sister 2b	SC:l,−2,3,−4,5,6,7,8	SC:1,3	4	64	-	-	-	4	2	2	0
Controls	-	SC:1,3,−4	4 (3)	32–64 (3)	32–64 (3)	32–64 (3)	16–32 (3)	4 (2)	4 (4)	2 (1)	0 (1)
Rd+ control	-	SC:1,3,4	2	32	NT	NT	NT	1	4	0	0

Abbreviation: -, not tested.

^aAll red cells were Fy(a-b+) and DAT negative; Radin (Sc4) testing not done in the patient and sister 2 because of ABO incompatibility.

^bHeterozygous for SCAR (Sc8) by sequencing.

4.4 |. Elution of anti-SCAR

Patientʼs plasma was incubated with Fy(a−b+) control red cells and an acid elution performed.²⁷ The eluate was positive with the control red cells, titer 1 (not shown). An anti-Sc3 was also adsorbed to the patientʼs and phenotypically matched control red cells, and the eluate was positive, titer 1 (not shown).

4.5 |. C3 complement activation

An anti-IgG agglutinated SC:8 red cells with 2+^s reactivity (score 9)²⁸ when sensitized by anti-SCAR. We did not detect C3b nor C3d on the sensitized red cells after incubation with freshly clotted AB serum as a source of complement (not shown).

4.6 |. MMA assay

The MMA phagocytic index for the anti-SCAR was <1, much below the threshold associated with clinically relevant alloantibodies (Table 4).

TABLE 4.

Monocyte monolayer assay (MMA) for anti-SCAR

	MMA phagocytic indexa		IATb
Red cell	Without complement	With complement	Without complement	With complement	Samples tested (n)
SCAR+ (SC:8)	0–0.1	0.1–0.4	2+	2+	3
SCAR− (SC:−8)	0.1	0.9	0	0	1
Controlc	55	-	4+	-	1

Abbreviation: -, not tested.

^aAn MMA phagocytic index of 5.0 or greater indicates clinical significance.

^bIndirect antiglobulin test (IAT): Saline method with polyclonal anti-human globulin in tubes.

^cPositive control: Polyclonal anti-D (Immucor) with R₂R₂ red cells.

4.7 |. IgG subclass

The anti-SCAR showed positive reactivity with the anti-IgG1. No reactivity was observed with anti-IgG2, anti-IgG3, or anti-IgG4.

4.8 |. Predicted effect of variants

Computational prediction by Franklin³⁶ indicated the ERMAP and RHCE substitutions to be either benign or of uncertain significance (Tables S1 and S2). Computational modeling by PredictSNP³⁸ also indicated the non-synonymous ERMAP:p.(Gln142Glu) as neutral with an expected accuracy of 83%.

4.9 |. Comparative homology modeling of the ERMAP protein

The template-based homology model of partial ERMAP protein comprised 150 amino acids from Lys36 to Leu186 including the Ig-like V-type domain (Val39 to Ala144). Based on our computer-generated 3D structure, there is a 37.4 Å distance between the C-alpha atoms of the amino acids at position 57 and 142 of the ERMAP protein (data not shown).

4.10 |. RHD and RHCE sequence

Zygosity testing by RFLP indicated two copies of the RHD gene in the proband and both sisters. Sequencing of RHD exon 7 (data not shown) and RHCE gene (Table S2) was compatible with homozygosity for an R²R² (DcE/DcE) genotype.

5 |. DISCUSSION

We defined the molecular basis of a high-prevalence antigen, named SCAR, in the Scianna blood group system. The SCAR antigen was identified in a Saudi Arabian patient with sickle cell beta thalassemia, who developed an alloantibody reactive in the indirect antiglobulin phase of testing with all red cells tested, except his own. The lack of the SCAR antigen was caused by homozygosity for a SNV (c.424C>G) in exon 4 of the ERMAP gene (Table 1). This pathognomonic variation, not found in the dbSNP database,⁴³ substitutes a glutamine in place of glutamic acid at position 142 of the ERMAP protein.

No transfusion reaction occurred and an MMA was negative; both observations suggested a limited clinical relevance of the anti-SCAR. The low titer of anti-SCAR in the patient (Table 3) may have prevented an obvious transfusion reaction at that time. Also, the MMA used allogeneic monocytes, which may not have closely replicated the immune status and Fc-receptor polymorphisms of the patientʼs monocytes.²⁰ However, the occurrence of anti-SCAR had a clinical consequence, because the patient was transitioned to hydroxyurea to prevent any boosting of the anti-SCAR titer through continued SCAR + red cell transfusions, which might eventually have resulted in a transfusion reaction.

Other alloantibodies to high-prevalence Scianna antigens, such as Sc3^21,44–46 and SCAN,²¹ have been documented to be clinically relevant.^7,13 Using autologous monocytes in an MMA has recently corroborated the clinical relevance of alloanti-Sc2 in a patient with severe hemolytic transfusion reaction.²⁰ Alloanti-Sc4 has also been known to cause HDFN.¹⁸ Of sufficient titer, all SC alloantibodies should be considered capable of causing hemolysis and HDFN.

The identification of SCAR expands the number of Scianna antigens to 8.⁷ The first antibody against an antigen of the Scianna blood group system was identified in 1958 (anti-Sc1),⁴⁷ described in 1962⁴⁷ and the antigenʼs molecular structure established in 2003;⁴⁸ while the seventh antibody (anti-SCAN) was identified in 1985,²¹ described in 1988,²¹ and the molecular basis established in 2005.⁴⁹ The anti-SCAR is the first new antibody found in the Scianna system during the previous 35 years.

The non-coding region of the 2 ERMAP alleles (MK933825 and MN013142) harbored multiple SNVs, which do not affect the expression of the ERMAP protein, as shown by serologic analyses (Table 3). All three individuals, the patient and his two sisters, were homozygous for an R₂R₂ phenotype (DDccEE), which has a 4% frequency in the Saudi Arabian population.⁵⁰ Both the ERMAP alleles (MK933825 and MN013142) identified in our study were linked to a normal R² (DcE) haplotype (Table 2). The intronic variations in the RHCE gene observed for this patient are known⁴³ (Table S2), and we have observed them previously (unpublished data). Previous studies documented that RH and SC alleles occur in a linkage disequilibrium although they are located 17.5 Mb apart.⁵¹

The ERMAP amino acid at position 142 is 85 amino acids away from residue 57, which distinguishes the anti-thetical antigens Sc1 and Sc2.⁴⁸ The serological results (Table 3) suggest that the amino acid variant 142Glu (SCAR+) does not affect expression of the high-prevalence antigen Sc1 (Gly57). According to Abbotts et al.,⁵² amino acids that are within 4 Å of each other are generally considered to be contacting residues. The C-alpha atoms of amino acids at position 57 and 142 are 37.4 Å apart in the three-dimensional ERMAP protein. Thus, they cannot be in direct contact with each other and are unlikely part of the same epitope. The above results support that both Sc1 and SCAR antigens are encoded by serologically independent epitopes. However, the positions might act as supporting residues required for proper conformation of the Ig-like V-type domain and epitope.

Being the first and only report of its occurrence, the prevalence of the rare SCAR-phenotype cannot be discerned. It may be limited to a specific ethnic group. To identify potential donors compatible with patients with anti-SCAR, the South Saudi Arabian population can be screened for SCAR− individuals. SCAR− red cells could also be grown ex vivo using autologous hematopoietic stem cells (HSCs).^53–56 Applications of in vitro generated red cells in transfusion⁵⁶ and as reagent^57,58 have been reviewed elsewhere.^59,60

High-prevalence antigens, such as SC:8, may not be considered clinically relevant enough for detection in current SNV-based genotyping platforms. However, their observations will be useful for analyzing large population wide datasets generated by next generation sequencing and determining their clinical relevance.