Blood group antigens arise from genetic polymorphisms and define numerous inter-individual differences, which can have tragic consequences in blood transfusions or pregnancy. Although the genetic locus of most blood group antigens has been identified (for recent reviews, see5,6
) with direct implications in transfusion medicine and obstetrics, the genetic basis remains unknown for some blood group antigens, despite their clinical significance. This is the case for the blood group antigen Lan, which can cause severe transfusion reactions7
as well as hemolytic disease of the fetus and newborn (HDFN)8,9
. Lan is a high-frequency antigen, not linked to any known blood group system, strongly suggesting that it is encoded by a new genetic locus. The Lan− blood type is very rare worldwide and seems inherited as a recessive trait7–10
. Lan-individuals are usually identified during serologic testing in order to investigate feto-maternal incompatibility and/or to find them compatible blood units, as they have developed an anti-Lan, i.e.
an alloantibody reacting with all red blood cells (RBCs) but Lan−. Transfusion support of patients with an anti-Lan is highly challenging due to the scarcity of compatible blood donors, but mainly due to the lack of reliable reagents for blood screening.
We have succeeded in generating the first monoclonal antibody with Lan specificity (OSK43, IgG1κ) from immortalized lymphocytes of a healthy Japanese Lan− woman who had developed an anti-Lan during pregnancy. OSK43 works in hemagglutination assays as well as flow cytometry and immunofluorescence analyses (), reacting with all tested RBCs but Lan−. We conducted a high-throughput screen by hemagglutination assay in Japan, and identified 14 Lan− persons in 713,384 blood donors, establishing a frequency of 0.002% for the Lan− blood type in this population. The intra- and inter-individual variation in RBC expression of the Lan antigen appeared notably low when evaluated by flow cytometry with OSK43 (). Nevertheless, we observed that OSK43 showed a higher reactivity with umbilical cord blood than adult RBCs () indicating that the Lan antigen expression can vary during development.
Characterization of the Lan blood group antigen expression on RBCs with the monoclonal antibody OSK43
The specificity and affinity of OSK43 suggested that it could be an important reagent to elucidate the genetic basis of the Lan blood group antigen. Towards this goal, we decided to use OSK43 to purify the Lan antigen-carrier protein from RBCs. As shown in , OSK43 was able to immunoprecipitate a RBC membrane protein of approximately 80 kDa, which was unambiguously identified as ABCB6 by mass spectrometry (). ABCB6 belongs to the large family of ABC transporters that use ATP to transport a wide variety of endogenous or xenobiotic substrates across cellular membranes (see11
). Based on phylogenetic analysis, ABCB6 has been classified in the sub-family B along with the multidrug resistance P-glycoprotein (P-gp aka ABCB1). ABCB6 has been described as a porphyrin transporter located in the outer mitochondrial membrane, highly expressed during erythroid differentiation and required for mitochondrial porphyrin uptake1
. Nevertheless, ABCB6 has also been found at the plasma membrane12
, consistent with its suspected role in multidrug resistance. Indeed, increased expression of ABCB6
correlated with increased resistance of different cancer cell lines to drugs2,3
, and ABCB6
copy number was increased in the camptothecin-resistant cell line A549/CPT4
. Thus, ABCB6 could transport anticancer drugs as well as endogenous substrates, as does ABCB1 and other multidrug ABC transporters (see13,14
Identification of ABCB6 as the carrier of the Lan blood group antigen
In order to validate ABCB6
as a novel genetic locus encoding blood group antigens, we first confirmed by western blot analysis that ABCB6 was present in the membrane of Lan+ RBCs (, top panel, lanes 3 and 6). In contrast, we detected no ABCB6
products in Lan− RBCs (, top panel, all other lanes), suggesting that null allele(s) of ABCB6
may be responsible for the Lan− blood type. We then established ABCB6
-expressing clones of the human cell line K-562 and analyzed them by flow cytometry with OSK43. We observed that exogenous expression of ABCB6
in K-562 cells correlated with cell surface expression of the Lan antigen (). Together, these results validate that the blood group antigen Lan is encoded by ABCB6
(2q36), thus defining a novel blood group system. Of note, 30 blood group systems are currently recognized by the International Society for Blood Transfusion, and none of them is encoded by an ABC transporter gene (http://ibgrl.blood.co.uk/isbtpages/isbtterminologypages/tableofbloodgroupsystems.htm
To identify the ABCB6
mutations responsible for the Lan− blood type, we used the Lan-blood samples cryopreserved in the rare blood collection of the National Reference Center for Blood Groups (Paris, France; the oldest sample dating to 1976). Of note, none of these Lan-blood samples was reactive with OSK43 by flow cytometry analysis (Supplementary Fig. 1
), confirming their original identification. We extracted genomic DNA from these blood samples and sequenced ABCB6
(see Supplementary Fig. 2
for sequencing strategy). From a cohort of 11 unrelated Lan− subjects, we identified 6 frameshift, 2 nonsense and 1 donor splice site mutations in ABCB6
(, mutations n°1 to 9, and Supplementary Fig. 3a–i
). Nine of these Lan-subjects were homozygous for a single mutation and two were double heterozygous (Supplementary Table 1
). Analysis of the available pedigrees confirmed that the Lan− blood type results from recessive inheritance of these ABCB6
mutations (Supplementary Fig. 4
). We also sequenced the genomic DNA of the Japanese Lan− woman whose lymphocytes were used to generate OSK43, and found that she was homozygous for yet another frameshift mutation in ABCB6
(, mutation n°10 and Supplementary Fig. 3j
), suggesting a wide variety of ABCB6
null alleles. All ten ABCB6
null mutations identified in this study were absent in control subjects (Lan+) and in NCBI dbSNP (build 132).
ABCB6 null mutations causing the Lan− blood type
Surprisingly, although ABCB6 was suspected to play an essential role in heme biosynthesis during erythroid differentiation by importing porphyrin into mitochondria1
) individuals did not exhibit anemia or abnormal erythropoiesis (Supplementary Fig. 5
). This unexpected finding indicates either that no transporter is actually required for porphyrin import into the mitochondrial inter-membrane space or, more likely, that another porphyrin transporter, yet unknown, can compensate for the absence of ABCB6 at the mitochondrial outer membrane. We show here that ABCB6 is present at the plasma membrane of RBCs, which are devoid of mitochondria in mammals, suggesting that ABCB6 could play a role in exporting porphyrin excess out of RBCs. Therefore, we measured blood levels of porphyrin in Lan-(ABCB6−/−
) individuals. Their RBC levels of porphyrin were slightly increased (2.1 µmol/L ± 0.2 (n=4), 0.1 < normal range < 1.9) but more strikingly, their plasma levels of porphyrin were very low and actually below the detection threshold (< 5.0 nmol/L (n=4), 6.5 < normal range < 20.0). While these findings were fully consistent with the absence of porphyria symptoms15
in Lan-individuals, they confirmed that ABCB6 is involved in porphyrin export from RBCs. However, the modest increase in RBC levels of porphyrin in ABCB6−/−
individuals suggests that another porphyrin transporter of the RBC membrane, such as the breast cancer resistance protein (BCRP aka ABCG2; , bottom panel)16
, may compensate for the absence of ABCB6 at the plasma membrane of RBCs. In fact, we show in an accompanying paper (NG-LE30370, Saison et al.
) that ABCG2−/−
individuals exhibit similarly impaired blood levels of porphyrin, indicating that ABCG2 and ABCB6 play a similar role in porphyrin export from RBCs. Yet, ABCG2 does not fully replace ABCB6 when the latter is absent, and vice versa
, otherwise ABCB6−/−
individuals would not exhibit impaired blood levels of porphyrin.
Since the Lan antigen is carried by ABCB6, and OSK43 is suitable for flow cytometry analysis of native cells, we decided to use OSK43 to evaluate cell surface expression of ABCB6 in different cell lines. As shown in , we found the Lan antigen on HepG2 hepatocellular carcinoma (HCC) cells, but not on A-498 renal cell carcinoma (RCC), A-431 squamous cell carcinoma (SCC), MOLT-4 acute lymphoblastic leukemia (ALL) or HeLa cervical cancer (CC) cells. When we examined another HCC cell line, HuH-7, we also detected the Lan antigen (). Expression of ABCB6 at the plasma membrane of HepG2 and HuH-7 cells may be either related to their hepatic origin, or acquired during hepatocarcinogenesis. Future studies will need to dissect the role of ABCB6 at the plasma membrane of human hepatocytes in health and disease. Nevertheless, it is worth mentioning that overexpression of ABCB6
has been observed in HCC compared to surrounding non-malignant tissue, and may contribute to multidrug resistance in HCC17
Expression of the Lan antigen on human cancer cell lines
In summary, we show in this report that ABCB6 is responsible for a novel blood group system, currently defined by the Lan antigen, which is present at the plasma membrane of RBCs but also HCC cells. A Lan-specific monoclonal antibody (OSK43) has been developed and will greatly facilitate the identification of Lan− blood donors as well as Lan− pregnant women, whose pregnancy is at risk of hemolytic disease of the fetus and newborn. By elucidating the genetic basis of the Lan− blood type, we have uncovered 10 null mutations of ABCB6 ( and ). No deficient alleles of ABCB6 have previously been reported and it was questionable whether complete deficiency of this porphyrin transporter was compatible with life. Not only is the complete deficiency of ABCB6 viable, but it is asymptomatic. Nevertheless, it will be necessary to closely monitor Lan− (ABCB6−/−) patients, especially those treated with drugs potentially transported by ABCB6, since its deficiency may alter pharmacokinetics of these drugs or result in adverse effects such as hepatotoxity.