• The inherited AnWj-negative blood group phenotype is caused by homozygosity for a deletion in MAL, encoding Mal protein.

  • Mal protein is expressed on red blood cell membranes of AnWj-positive, but not AnWj-negative, individuals.

Abstract

The genetic background of the high prevalence red blood cell antigen AnWj has remained unresolved since its identification in 1972, despite reported associations with both CD44 and Smyd1 histone methyltransferase. Development of anti-AnWj, which may be clinically significant, is usually due to transient suppression of antigen expression, but a small number of individuals with persistent, autosomally recessive inherited AnWj-negative phenotype have been reported. Whole-exome sequencing of individuals with the rare inherited AnWj-negative phenotype revealed no shared mutations in CD44H or SMYD1; instead, we discovered homozygosity for the same large exonic deletion in MAL, which was confirmed in additional unrelated AnWj-negative individuals. MAL encodes an integral multipass membrane proteolipid, myelin and lymphocyte protein (Mal), which has been reported to have essential roles in cell transport and membrane stability. AnWj-positive individuals were shown to express full-length Mal on their red cell membranes, which was not present on the membranes of AnWj-negative individuals, regardless of whether from an inherited or suppression background. Furthermore, binding of anti-AnWj was able to inhibit binding of anti-Mal to AnWj-positive red cells, demonstrating the antibodies bind to the same molecule. Overexpression of Mal in an erythroid cell line resulted in the expression of AnWj antigen, regardless of the presence or absence of CD44, demonstrating that Mal is both necessary and sufficient for AnWj expression. Our data resolve the genetic background of the inherited AnWj-negative phenotype, forming the basis of a new blood group system, further reducing the number of remaining unsolved blood group antigens.

1.
International Society of Blood Transfusion (ISBT)
.
Red cell immunogenetics and blood group terminology
. Accessed 22 February 2024. https://www.isbtweb.org/isbt-working-parties/rcibgt.html.
2.
Marsh
WL
,
Brown
PJ
,
DiNapoli
J
, et al
.
Anti-Wj: an autoantibody that defines a high-incidence antigen modified by the In(Lu) gene
.
Transfusion
.
1983
;
23
(
2
):
128
-
130
.
3.
Boorman
KE
,
Tippett
P
. Unpublished observations.
Blood groups in man
.
Blackwell
;
1975
:
274
-
275
.
4.
Poole
J
,
Van Alphen
L
.
Haemophilus influenzae receptor and the AnWj antigen
.
Transfusion
.
1988
;
28
(
3
):
289
.
5.
Poole
J
,
Giles
CM
.
Observations on the Anton antigen and antibody
.
Vox Sang
.
1982
;
43
(
4
):
220
-
222
.
6.
Alphen
L
,
Poole
J
,
Overbeeke
M
.
The Anton blood group antigen is the erythrocyte receptor for Haemophilus influenzae
.
FEMS Microbiol Lett
.
1986
;
37
(
1
):
69
-
71
.
7.
Singleton
BK
,
Burton
NM
,
Green
C
,
Brady
RL
,
Anstee
DJ
.
Mutations in EKLF/KLF1 form the molecular basis of the rare blood group In(Lu) phenotype
.
Blood
.
2008
;
112
(
5
):
2081
-
2088
.
8.
Taliano
V
,
Guévin
R-M
,
Tippett
P
.
The genetics of a dominant inhibitor of the Lutheran antigens
.
Vox Sang
.
1973
;
24
(
1
):
42
-
47
.
9.
Spring
FA
,
Dalchau
R
,
Daniels
GL
, et al
.
The Ina and Inb blood group antigens are located on a glycoprotein of 80 000 MW (the CDw44 glycoprotein) whose expression is influenced by the In(Lu) gene
.
Immunology
.
1988
;
64
(
1
):
37
-
43
.
10.
Mannessier
L
,
Rouger
P
,
Johnson
CL
,
Mueller
KA
,
Marsh
WL
.
Acquired loss of red-cell Wj antigen in a patient with Hodgkin's disease
.
Vox Sang
.
1986
;
50
(
4
):
240
-
244
.
11.
Harris
T
,
Steiert
S
,
Marsh
WL
,
Berman
LB
.
A Wj-negative patient with anti-Wj
.
Transfusion
.
1986
;
26
(
1
):
117
.
12.
de Man
AJ
,
van Dijk
BA
,
Daniels
GL
.
An example of anti-AnWj causing haemolytic transfusion reaction
.
Vox Sang
.
1992
;
63
(
3
):
238
.
13.
Xu
Z
,
Duffett
L
,
Tokessy
M
,
Cote
J
,
Goldman
M
,
Saidenberg
E
.
Anti-AnWj causing acute hemolytic transfusion reactions in a patient with aplastic anemia
.
Transfusion
.
2012
;
52
(
7
):
1476
-
1481
.
14.
Poole
J
,
Levene
C
,
Bennett
M
,
Sela
R
,
van Alphen
L
,
Spruell
PJ
.
A family showing inheritance of the Anton blood group antigen AnWj and independence of AnWj from Lutheran
.
Transfus Med
.
1991
;
1
(
4
):
245
-
251
.
15.
Yong
J
,
Johnstone
M
,
Callaghan
T
,
Chandrasekar
A
,
Pervaiz
MS
.
Anti-AnWj antibody induced haemolytic transfusion reaction in a patient with primary acquired pure red cell aplasia
.
Transfus Med
.
2022
;
32
(
4
):
346
-
348
.
16.
Telen
M
,
Rao
N
,
Udani
M
,
Liao
H-X
,
Haynes
BF
.
Relationship of the AnWj blood group antigen to expression of CD44 [abstract]
.
Transfusion
.
1993
;
33
(
suppl
):
48
. Abstract S182.
17.
Rao
N
,
Udani
M
,
Telen
MJ
.
Demonstration by monoclonal antibody immobilization of erythrocyte antigens and dot blot that both the In and AnWj blood group antigens reside on CD44
.
Transfusion
.
1994
;
34
(
suppl
):
25
. Abstract S99.
18.
Yahalom
V
,
Pillar
N
,
Zhao
Y
, et al
.
SMYD1 is the underlying gene for the AnWj-negative blood group phenotype
.
Eur J Haematol
.
2018
;
101
(
4
):
496
-
501
.
19.
Puertollano
R
,
Alonso
MA
.
Targeting of MAL, a putative element of the apical sorting machinery, to glycolipid-enriched membranes requires a pre-golgi sorting event
.
Biochem Biophys Res Commun
.
1999
;
254
(
3
):
689
-
692
.
20.
Alonso
MA
,
Weissman
SM
.
cDNA cloning and sequence of MAL, a hydrophobic protein associated with human T-cell differentiation
.
Proc Natl Acad Sci USA
.
1987
;
84
(
7
):
1997
-
2001
.
21.
Rancaño
C
,
Rubio
T
,
Alonso
MA
.
Alternative splicing of human T-cell-specific MAL mRNA and its correlation with the exon/intron organization of the gene
.
Genomics
.
1994
;
21
(
2
):
447
-
450
.
22.
Zacchetti
D
,
Peränen
J
,
Murata
M
,
Fiedler
K
,
Simons
K
.
VIP17/MAL, a proteolipid in apical transport vesicles
.
FEBS Lett
.
1995
;
377
(
3
):
465
-
469
.
23.
Kim
T
,
Fiedler
K
,
Madison
DL
,
Krueger
WH
,
Pfeiffer
SE
.
Cloning and characterization of MVP17: a developmentally regulated myelin protein in oligodendrocytes
.
J Neurosci Res
.
1995
;
42
(
3
):
413
-
422
.
24.
Schaeren-Wiemers
N
,
Valenzuela
DM
,
Frank
M
,
Schwab
ME
.
Characterization of a rat gene, rMAL, encoding a protein with four hydrophobic domains in central and peripheral myelin
.
J Neurosci
.
1995
;
15
(
8
):
5753
-
5764
.
25.
Puertollano
R
,
Martín-Belmonte
F
,
Millán
J
, et al
.
The MAL proteolipid is necessary for normal apical transport and accurate sorting of the influenza virus hemagglutinin in Madin-Darby canine kidney cells
.
J Cell Biol
.
1999
;
145
(
1
):
141
-
151
.
26.
Geng
Z
,
Huang
J
,
Kang
L
, et al
.
Clostridium perfringens epsilon toxin binds to erythrocyte MAL receptors and triggers phosphatidylserine exposure
.
J Cell Mol Med
.
2020
;
24
(
13
):
7341
-
7352
.
27.
Lara-Lemus
R
.
On the role of myelin and lymphocyte protein (MAL) in cancer: a puzzle with two faces
.
J Cancer
.
2019
;
10
(
10
):
2312
-
2318
.
28.
Mimori
K
,
Shiraishi
T
,
Mashino
K
, et al
.
MAL gene expression in esophageal cancer suppresses motility, invasion and tumorigenicity and enhances apoptosis through the Fas pathway
.
Oncogene
.
2003
;
22
(
22
):
3463
-
3471
.
29.
Lind
GE
,
Ahlquist
T
,
Kolberg
M
, et al
.
Hypermethylated MAL gene - a silent marker of early colon tumorigenesis
.
J Transl Med
.
2008
;
6
:
13
.
30.
Kalmár
A
,
Péterfia
B
,
Hollósi
P
, et al
.
DNA hypermethylation and decreased mRNA expression of MAL, PRIMA1, PTGDR and SFRP1 in colorectal adenoma and cancer
.
BMC Cancer
.
2015
;
15
:
736
.
31.
Horne
HN
,
Lee
PS
,
Murphy
SK
,
Alonso
MA
,
Olson
JA
,
Marks
JR
.
Inactivation of the MAL gene in breast cancer is a common event that predicts benefit from adjuvant chemotherapy
.
Mol Cancer Res
.
2009
;
7
(
2
):
199
-
209
.
32.
Blaveri
E
,
Simko
JP
,
Korkola
JE
, et al
.
Bladder cancer outcome and subtype classification by gene expression
.
Clin Cancer Res
.
2005
;
11
(
11
):
4044
-
4055
.
33.
Kohno
T
,
Moriuchi
R
,
Katamine
S
,
Yamada
Y
,
Tomonaga
M
,
Matsuyama
T
.
Identification of genes associated with the progression of adult T cell leukemia (ATL)
.
Jpn J Cancer Res
.
2000
;
91
(
11
):
1103
-
1110
.
34.
Copie-Bergman
C
,
Gaulard
P
,
Maouche-Chrétien
L
, et al
.
The MAL gene is expressed in primary mediastinal large B-cell lymphoma
.
Blood
.
1999
;
94
(
10
):
3567
-
3575
.
35.
Hsi
ED
,
Sup
SJ
,
Alemany
C
, et al
.
MAL is expressed in a subset of Hodgkin lymphoma and identifies a population of patients with poor prognosis
.
Am J Clin Pathol
.
2006
;
125
(
5
):
776
-
782
.
36.
Zanotti
L
,
Romani
C
,
Tassone
L
, et al
.
MAL gene overexpression as a marker of high-grade serous ovarian carcinoma stem-like cells that predicts chemoresistance and poor prognosis
.
BMC Cancer
.
2017
;
17
(
1
):
366
.
37.
Lee
PS
,
Teaberry
VS
,
Bland
AE
, et al
.
Elevated MAL expression is accompanied by promoter hypomethylation and platinum resistance in epithelial ovarian cancer
.
Int J Cancer
.
2010
;
126
(
6
):
1378
-
1389
.
38.
Elpidorou
M
,
Poulter
JA
,
Szymanska
K
, et al
.
Missense mutation of MAL causes a rare leukodystrophy similar to Pelizaeus-Merzbacher disease
.
Eur J Hum Genet
.
2022
;
30
(
7
):
860
-
864
.
39.
Judd
JW
,
Johnson
ST
,
Storry
J
. Judd’s methods in immunohematology. 3rd ed..
AABB Press
;
2008
.
40.
Knowles
RW
,
Bai
Y
,
Lomas
C
,
Green
C
,
Tippett
P
.
Two monoclonal antibodies detecting high frequency antigens absent from red cells of the dominant type of Lu(a-b-) Lu:-3. 1982
.
J Immunogenet
.
1982
;
9
(
5
):
353
-
357
.
41.
Thorvaldsdóttir
H
,
Robinson
JT
,
Mesirov
JP
.
Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. 2013
.
Brief Bioinform
.
2013
;
14
(
2
):
178
-
192
.
42.
Dodge
JT
,
Mitchell
C
,
Hanahan
DJ
.
The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes
.
Arch Biochem Biophys
.
1963
;
100
:
119
-
130
.
43.
Millán
J
,
Alonso
MA
.
MAL, a novel integral membrane protein of human T lymphocytes, associates with glycosylphosphatidylinositol-anchored proteins and Src-like tyrosine kinases
.
Eur J Immunol
.
1998
;
28
(
11
):
3675
-
3684
.
44.
Thornton
N
,
Karamatic Crew
V
,
Tilley
L
, et al
.
Disruption of the tumour-associated EMP3 enhances erythroid proliferation and causes the MAM-negative phenotype
.
Nat Commun
.
2020
;
11
(
1
):
3569
.
45.
Karamatic Crew
V
,
Tilley
LA
,
Satchwell
TJ
, et al
.
Missense mutations in PIEZO1, which encodes the Piezo1 mechanosensor protein, define Er red blood cell antigens
.
Blood
.
2023
;
141
(
2
):
135
-
146
.
46.
Bryk
AH
,
Wiśniewski
JR
.
Quantitative analysis of human red blood cell proteome
.
J Proteome Res
.
2017
;
16
(
8
):
2752
-
2761
.
47.
Ravenhill
BJ
,
Kanjee
U
,
Ahouidi
A
, et al
.
Quantitative comparative analysis of human erythrocyte surface proteins between individuals from two genetically distinct populations
.
Commun Biol
.
2019
;
2
:
350
.
48.
Rauluseviciute
I
,
Riudavets-Puig
R
,
Blanc-Mathieu
R
, et al
.
JASPAR 2024: 20th anniversary of the open-access database of transcription factor binding profiles
.
Nucleic Acids Res
.
2024
;
52
(
D1
):
D174
-
D182
.
49.
Lee
E
,
Burgess
G
,
Win
N
.
Autoimmune hemolytic anemia and a further example of autoanti-Kpb
.
Immunohematology
.
2005
;
21
(
3
):
119
-
121
.
50.
Issitt
PD
,
Obarski
G
,
Hartnett
PL
,
Wren
MR
,
Prewitt
PL
.
Temporary suppression of Kidd system antigen expression accompanied by transient production of anti-Jk3
.
Transfusion
.
1990
;
30
(
1
):
46
-
50
.
51.
Giles
CM
,
Lundsgaard
AA
.
A complex serological investigation involving LW
.
Vox Sang
.
1967
;
13
(
5
):
406
-
416
.
52.
Chown
B
,
Kaita
H
,
Lowen
B
,
Lewis
M
.
Transient production of anti-LW by LW-positive people
.
Transfusion
.
1971
;
11
(
4
):
220
-
222
.
53.
Parsons
SF
,
Jones
J
,
Anstee
DJ
, et al
.
A novel form of congenital dyserythropoietic anemia associated with deficiency of erythroid CD44 and a unique blood group phenotype [In(a-b-), Co(a-b-)]
.
Blood
.
1994
;
83
(
3
):
860
-
868
.
54.
Singleton
BK
,
Fairweather
VSS
,
Lau
W
, et al
.
A novel EKLF mutation in a patient with dyserythropoietic anemia: the first association of EKLF with disease in man [abstract]
.
Blood
.
2009
;
114
(
22
):
162
.
55.
Arnaud
L
,
Saison
C
,
Helias
V
, et al
.
A dominant mutation in the gene encoding the erythroid transcription factor KLF1 causes a congenital dyserythropoietic anemia
.
Am J Hum Genet
.
2010
;
87
(
5
):
721
-
727
.
56.
Tugores
A
,
Rubio
T
,
Rancaño
C
,
Alonso
MA
.
A tandem array of Sp-1 sites and a reverse initiator element are both required for synergistic transcriptional activation of the T-cell-specific MAL gene
.
DNA Cell Biol
.
1997
;
16
(
3
):
245
-
255
.
57.
Langley
SA
,
Miga
KH
,
Karpen
GH
,
Langley
CH
.
Haplotypes spanning centromeric regions reveal persistence of large blocks of archaic DNA
.
Elife
.
2019
;
8
:
e42989
.
58.
Miller
LH
,
Mason
SJ
,
Clyde
DF
,
McGinniss
MH
.
The resistance factor to Plasmodium vivax in blacks. The Duffy-blood-group genotype, FyFy
.
N Engl J Med
.
1976
;
295
(
6
):
302
-
304
.
59.
Miller
LH
,
Mason
SJ
,
Dvorak
JA
,
McGinniss
MH
,
Rothman
IK
.
Erythrocyte receptors for (Plasmodium knowlesi) malaria: Duffy blood group determinants
.
Science
.
1975
;
189
(
4202
):
561
-
563
.
60.
Van Alphen
L
,
Levene
C
,
Geelen-van den Broek
L
,
Poole
J
,
Bennett
M
,
Dankert
J
.
Combined inheritance of epithelial and erythrocyte receptors for Haemophilus influenzae
.
Infect Immun
.
1990
;
58
(
11
):
3807
-
3809
.
61.
Gao
J
,
Xin
W
,
Huang
J
, et al
.
Hemolysis in human erythrocytes by Clostridium perfringens epsilon toxin requires activation of P2 receptors
.
Virulence
.
2018
;
9
(
1
):
1601
-
1614
.
62.
Rumah
KR
,
Ma
Y
,
Linden
JR
, et al
.
The myelin and lymphocyte protein MAL is required for binding and activity of Clostridium perfringens ε-toxin
.
PLoS Pathog
.
2015
;
11
(
5
):
e1004896
.
63.
Rumah
KR
,
Linden
J
,
Fischetti
VA
,
Vartanian
T
.
Isolation of Clostridium perfringens type B in an individual at first clinical presentation of multiple sclerosis provides clues for environmental triggers of the disease
.
PLoS One
.
2013
;
8
(
10
):
e76359
.
64.
Novitzky-Basso
I
,
Spring
F
,
Anstee
D
,
Tripathi
D
,
Chen
F
.
Erythrocytes from patients with myeloproliferative neoplasms and splanchnic venous thrombosis show greater expression of Lu/BCAM
.
Int J Lab Hematol
.
2018
;
40
(
4
):
473
-
477
.
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