Hybrid glycophorin GP (A-B) and anti-En^a antibody in a multiparous woman

Abstract

The MNS system is a large, complex group of antigens, with 50 antigens at present. The major antigens of the system are carried on glycophorins A and B, which are important red cell sialoglycoproteins. Altered expression of MNS antigens is possible in cases where there is a formation of hybrid glycophorins. Some of these patients may make clinically significant antibodies which can be associated with hemolytic transfusion reactions (HTR) or hemolytic disease of the fetus and the newborn. This is a report of one such case with both these phenomena, in a multiparous woman. Incompatible cross-matches at the referral center caused the samples to be sent to this immunohematological laboratory where the serological profile was detailed. On sending the samples to a third blood group reference laboratory, gene sequencing was done and the antibody was identified. At this immunohematological laboratory, the phenotype was determined as M-N+. The patient’s sera showed panreactivity with all panel cells (3-cell and 11-cell). At blood group reference laboratory, the findings were as follows- GYP (A-B) hybrid gene consistent with hemi/homozygosity for GYP*JL was confirmed and the panreactive antibody was identified as anti - En^a. Rare, atypical phenotypes in the MNS system can result from mutations affecting the sialoglycoproteins the antigens are carried on. The subset of antigens expressed in individual phenotypes varies and these patients can develop antibodies against the antigens they lack. When they present with immunohematological challenges such as panreactivity and incompatibility, a combination of serology, molecular technologies, and family studies help solve the problem. Alternatives to transfusion are also useful options in managing such cases.

Introduction

The MNS system (ISBT 002) is the second blood group system to be discovered and is incredibly complex. The most well-described antigens of the system, M and N are antithetical to each other and differ in their amino acid structures at two positions. Another set of antithetical antigens in the system S and s varies by one amino acid only. Red cell sialoglycoproteins (GYPA and GYPB) carry the major antigens of the system and antibodies against these antigens may be clinically significant when reactive at 37°C.[1]

The absence of portions or complete segments of one or both of these glycoproteins prevents expression of the antigens they carry. The most prominent examples are the En (a-) and M^k phenotypes. Another category of atypical phenotypes may also result from hybrid glycophorins. These hybrids are formed due to phenomena such as substitution, gene conversion, deletion, and crossing over of genes on chromosomes.[2]

All these phenotypes are associated with expressions of different subsets of antigens in the system and may develop antibodies against the missing antigens, making compatibility testing challenging. This report describes a case of a rare MNS phenotype and the steps that led to its detection.

Case Report

At the parent center, a 36-year-old woman with the obstetric profile of P2 L2 with a fibroid uterus was being evaluated for possible surgery. She had a history of transfusion of 3 units of packed red cells during two previous hospital admissions, which had been approximately 24 and 12 months before this current visit. She had reportedly developed jaundice after her last transfusion. No further information could be elicited about the transfusion or the reaction. When trying to arrange blood for her for possible use during her surgery, all units were found to be incompatible. At this point, her sample was sent to our immunohematological reference laboratory for further assessment.

At the immunohematological reference laboratory

The ABO grouping and RhD typing were done by column agglutination technology (CAT) using gelcards (ID-Micro Typing system A/B/D monoclonal and reverse grouping card) in the Ortho vision swift analyzer (both from Ortho Clinical Diagnostics, Raritan, New Jersey, USA). The blood group was found to be O Positive. Direct antiglobulin test and autocontrol were done by CAT on gelcards (ID-Micro typing System Anti-IgG, C3d card) and were negative. When indirect antiglobulin test (IAT), done by CAT on gelcard (ID-Micro typing System Anti-IgG, C3d card, Ortho Clinical Diagnostics, Raritan, New Jersey, USA) using in-house pooled O cells was found to be (3+) positive, it was followed up with 3-cell antibody screening and then 11-cell antibody identification (reagent red blood cells – 0.8% Surgiscreen and Resolve Panel C system, respectively, Ortho Clinical Diagnostics, Raritan, New Jersey, USA) by CAT (ID-Micro typing System Anti-IgG, C3d card, Ortho Clinical Diagnostics, Raritan, New Jersey, USA) to yield 3+ panreactivity with all the cells. The potentiator used was low ionic strength saline (LISS, Ortho Clinical Diagnostics, Raritan, New Jersey, USA) [Figures 1-3]. Antibody screening and identification were also repeated by conventional tube testing method through three stages (Immediate spin/room temperature, 37°C, antihuman globulin [AHG] phase) with the same commercial cells used for the CAT. 3+ reaction was seen with all cells, only at the AHG phase.

Figure 1.

Gel card (CAT, ortho clinical diagnostics) photograph of 3-cell antibody screen (lot number 3SS923) and autocontrol, performed on the Ortho Vision Swift Analyzer: Cell I, II and III- 3+, autocontrol- Negative (Page 3)

Figure 3.

Gel card (CAT, ortho clinical diagnostics) photograph of 11-cell antibody identification (lot number RA153), performed on the Ortho Vision Swift Analyzer: Cell 1-11- 3+ (Page 3)

Figure 2.

Antigram for 3-cell antibody screening (lot number 3SS923) by CAT (ortho clinical diagnostics) and antigen phenotyping (Tulip Diagnostics, Goa), performed on the Ortho Vision Swift Analyzer: Cell I, II and III- 3+, C+E-c-e+K-k+Fya+Fyb-Jka+Jkb+Lea-Leb+S+s-M-N+ (Page 3)

To evaluate this panreactivity, Anti-H lectin (Ortho Clinical Diagnostics, Raritan, New Jersey, USA) test was done (4+) and extended RBC antigen phenotyping by CAT by gelcards (ID-Micro typing System Anti-IgG, C3d card, Ortho Clinical Diagnostics, Raritan, New Jersey, USA) and respective antisera (Ortho Clinical Diagnostics, Raritan, New Jersey, USA) showed her phenotype as C+E-c-e+K-k+Fy^a+Fy^b-Jk^a+Jk^b+M-N+S+s-Le^a-Le^b+.

Suspecting an antibody to a high frequency antigen, the sample was sent to the International Blood Group Reference Laboratory (IBGRL), Bristol for genotyping and further serological work-up.

At IBGRL: The report from this laboratory included the following major findings:

• THE ANTIBODY: Eluate was prepared from antigen-matched cells (c-E-s-K-Fy (b)-) following adsorption with the patient’s serum. It was compatible with En (a-) and M^kM^k cells and incompatible with cells of phenotype Wr (a + b-) En (a+). Hence, the presence of anti-En^a antibody was suggested. It reacted strongly by LISS IAT with untreated and papain-treated cells. The only cells found to be compatible with the patient’s serum were an example of M^kM^k cells and two examples of En (a-) cells

• Antibodies ruled out: c, E, s, Fy^b. Anti-K and Anti-Wr^a could not be ruled out

• THE PHENOTYPE: Genotypic sequencing (3500 XL Genetic Analyzer for human identification, Thermo Fisher Scientific, Waltham, Massachusetts) of the patient’s cells for GYPA and GYPB showed the presence of N (exon 2 of GYPA) and S (exon 4 of GYPB) antigens. Long-range polymerase chain reaction (PCR) amplification (C1000 Touch Thermal Cycler, BioRad, Cressier, Switzerland) of both of those genes showed a hybrid GYP A-B, consistent with homo (or hemi) zygosity for GYP*JL. It encodes En (a-) Wr (a-b-) TSEN+MINY+ [Figure 4].

Figure 4.

An extract from the sequencing report from IBGRL showing the possible mutation in the patient (Page 4)

In view of her complicated immunohematological profile, her physicians were informed of the findings and advised hematinics and possible autologous transfusion if necessary. Family studies were also advised but could not be coordinated due to logistical difficulties from the patient’s end. She was monitored throughout her pregnancy. Fortunately, it progressed uneventfully and she delivered a healthy term baby.

Discussion

The MNS system, with 50 antigens, is an extremely complex blood group antigen system. The major antigens are carried on sialoglycoproteins called Glycophorin A (GPA) and glycophorin B (GPB). Normally, the N-terminal peptide of GPA carries the M/N antigen, and the C-terminal carries the S/s antigen. M/N and S/s are antithetical antigens. Single nucleotide polymorphisms at amino acids 20 and 24 of GPA and 48 of GPB determine which one antigen in each set will be expressed.[1]

Genetic mutations leading to the absence of these sialoglycoproteins also cause an absence of the antigens they carry. These patients may then develop antibodies against the antigens they are lacking.

Rare phenotypes have also been described due to hybrid glycophorins.[1] Owing to genetic phenomena such as misalignment and unequal crossing over, novel fusion genes are formed that code for hybrid molecules such as GP (A-B). Here, the N-terminal region is from the GPA and the C-terminal region is from the GPB. GP (B-A), GP (B-A-B), and GP (A-B-A) have also been described. The expression of the MNS antigens also changes according to the mutation.[1]

The now obsolete Miltenberger series is a series of rare MNS phenotypes, related to each other through the overlapping specificities of some low-frequency alloantigens. It is based on the serological pattern of reaction of the typing sera in the cells in question. The eleventh class of this series (Class XI), notified as GP*JL, has cells reacting with anti-TSEN and anti-MINY. This is a GP (A-B) hybrid which has exons A1-A3 of GYPA fused with exons B4-B6 of GYPB. This equates to amino acids 1–58 from GPA and 27 to 72 of GPB. Methionine instead of threonine at position 61 gives it an S activity. Previously reported GP*JL cases have also produced anti-En^a and/or anti-Wr^b. They carry TSEN and MINY antigens.[1]

The anti-En^a antibodies may develop in individuals due to a homozygous deletion in the GYPA causing no production of GPA or in GP (A-B) hybrid phenotypes. They react with all cells except those of En (a-) and M^kM^k phenotype. These are clinically significant antibodies capable of causing hemolytic disease of the newborn and HTR. A similar case report describes a patient heterozygous for GYP. JL and M^k with an alloantibody to a high-prevalence antigen, were identified to be anti-En^aFR.[3] Anti-En^a is an umbrella term used to denote antibodies reactive against various portions of the GPA, unrelated to M or N, but individual antibodies detect different portions. Anti-En^aFR detects a ficin-resistant area between amino acids 62 and 72.[2]

This patient was a multiparous woman with a history suggestive of prior transfusion reaction, showing panreactivity with all cells equally in the screening and identification panels. This made compatibility testing challenging. Her MNS phenotype, as confirmed by serology and genetic testing, showed the presence of N and S antigens. PCR and genetic sequencing also reported hemi-/homozygosity for the hybrid GP (A-B) phenotype [Figure 4] consistent with the variant archived in the ISBT resources,[4] i.e., the GYP*JL (GYP*202.01). The antibody was identified as anti-En (a-). A similar profile was seen in a Thai patient also demonstrating an anti-En (a-) antibody with homozygosity for GYPA*M c.295delG (p.Val99Ter). His phenotype was described with the help of DNA sequencing and serology during the investigation of a HTR.[5]

In these kinds of findings, the possible next step can be to detect and quantify the glycophorins and the antigens concerned. To this end, a plethora of other techniques such as flow cytometry and immunoblotting have been used in other studies.[6] A Chinese study, aiming to establish the correlation between alternative RNA splicing and the antigenic features and cell surface distribution of the glycophorins found that RNA splicing affects the former but not the latter. They used and suggested mRNA analysis as a valuable supplement to DNA sequencing and serology.[7] However, in our case, none of these was possible due to logistical and financial constraints.

Once the antibody is identified, the clinical management side of the patient care has been outlined in another case report of anti-En (a-) in a pregnant woman.[8] In our case, the pregnancy was uneventful and required no additional interventions with respect to her blood needs.

Conclusion

Complex RBC antigen systems like MNS can have a lot of rare, atypical phenotypes which present with routine laboratory phenomena such as panreactive sera or cross-match incompatibility. Standard serological methods, backed by clinical history and if needed, advanced immunohematological techniques and PCR sequencing are imperative in solving such cases. Alternative approaches such as hematinics and autologous transfusion should be considered to reduce and meet blood needs in such patients.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient has given her consent for her images and other clinical information to be reported in the journal. The patient understands that her name and initials will not be published and due efforts will be made to conceal her identity, but anonymity cannot be guaranteed.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.