(p-ig-4) pigg defines the emm blood group system
TRANSCRIPT
William J. Lane1,2, Judith Aeschlimann3, Sunitha Vege3, Christine Lomas-Francis3, Anna Burgos3, Helen H. Mah1, Justin Halls1,2, Peter C. Ligthart4, Barbera Veldhuisen4, Ripal J. Shah5, Sanmukh R. Joshi6, Connie M. Westhoff3
1Department of Pathology, Brigham and Women’s Hospital, 75 Francis St, Boston, MA. 2Harvard Medical School, 25 Shattuck St, Boston, MA. 3New York Blood Center, 310 E 67th St, New York, NY. 4Department of Immunohematology Diagnostic Services, Sanquin, Amsterdam, Netherlands. 5Prathama Blood Center, Ahmedabad, India. 6Department of Research, Lok Samarpan Raktdan Kendra, Surat, India.
BackgroundEmm is one of the few remaining blood groups whose basis is unknown. It is a high incidence red cell antigen with eight Emm− probands reported over the past 30 years. Anti-Emm appears to be naturally occurring yet responsible for a clinically significant acute hemolytic transfusion reaction. Previous work showed that Emm is located on a GPI-anchored protein, but the antigenic epitope and genetic basis have been elusive.
PIGG Defines the Emm Blood Group System
Whole Genomes Sequencing (WGS) of an Emm− Proband and family revealed a PIGG loss of function variant.PIGG loss of function variants found in four other probands. PIGG adds EtNP onto GPI-anchor.
Identification of PIGG Deficiency In Emm− Proband 1
For more details see our recent paperLane WJ, et al. Sci Rep. 2021 Sep 17;11(1):18545.doi: 10.1038/s41598-021-98090-w. PMID: 34535746.https://bit.ly/3ooLHIl
(A) Family Pedigree. Emm RBC phenotypes shown include the Proband and his brother, both with anti-Emm in the plasma, the Probands spouse and two children who are Emm+ and were shown to lack the antibody. (B) Study Outline. Emm serologic RBC typing was performed with in-house reagents and short read whole genome sequencing (WGS). (C) Variant Identification. Illustration of the strategy used to enrich for loss of function mutations associated with familial inheritance and variant effector prediction (VEP) scoring to identify the genetic cause of the Emm− phenotype. PIGG was the only candidate gene that passed our filtering strategy. (D) WGS alignments. IGV genomics viewer shows the wild type sequence (upper) and PIGG Exon 12 biallelic sequence for each family member (below). Individual sequence reads are shown in gray for positions corresponding to the hg38 reference sequence with bar plots above (dark grey) reflecting the relative number of reads at that position. Emm− family members were homozygous for a 2-bp deletion in PIGG Exon 12, predicted to cause a frameshift and premature stop, designated in as c.2624_2625delTA, p.(Leu875*), rs771819481, and chromosomal location hg38:chr4:533870_533871delTA. The spouse was wild type, and the daughter and son are heterozygous. The following PIGGreference sequences were used throughout the manuscript: NG_051621.1 (genomic) and NM_001127178.3 (transcript)
B
Figure 2
PIGG Exons 1-13
C Proband 2
A Proband 1
Spouse (Emm+)
Control (Emm+)
Daughter (Emm+)
Son (Emm+)
PIGG Exons
PIGG Exons 1-4Long Range PCR
1 2 3 4
PIGG Exons PIGG Exons
Heterozygous c.2624_2625delTAHomozygous c.2624_2625delTAWild TypeProband 1 (Emm−)
Proband 2 (Emm−) Proband 3 (Emm−)
TCGTGGGCTTAGACACCTACTCGTGGGCTTAGACACCTAC
Brother (Emm−)
Control (Emm+)
PIGG Exon 12
chr4:500,000
500040003000200015001000
700500400300200
75
70001000020000
(bp)
502,000
Con
trol
Prob
and-
2
NG
S Pa
ired
Split
Rea
dsSa
nger
PIGG Exons
504,000 506,000
Intron 1
Intron 1
Intron 3
Intron 3Ta
rget
ed N
GS
6.0 kb deletion (Exons 2-3) CTGAGGATAATTCACCCAAAGCTTAGAACACTGAGGATAATTCACCCAAAGCTTAGAACA
NGS Breakpoint Breakpoint Sequence
Contro
l
Φ Proba
nd 2
No DNA
Cont
rol
Φ Prob
and
1
No D
NA
Son
Daug
hter
Spou
se
Brot
her
paired split reads spanning deletion
10 13Φ 1&2 3 4 5 6 7&8 9 1211 10 13Φ 1&2 3 4 5 6 7&8 9 1211 10 13Φ 1&2 3 4 5 6 7&8 9 1211
700600500400300200100
800900
1000(bp)
700600500400300200100
800900
1000(bp)
TCGTGGGCTGACACCTACTCGTGGGCTGACACCTAC
TCGTGGGCTTAGACACCTACTCGTGGGCTTAGACACCTAC
TCGTGGGCTGACACCTACTCGTGGGCTGACACCTAC
TCGTGGGCTTAGACACCTACTCGTGGGCTGACACCTACGT
TCGTGGGCTTAGACACCTACTCGTGGGCTGACACCTACGT
Left
Rig
ht
C T G A G G A T A A T T C A C C C A A A G C T T A G A A C AC T G A G G A T A A T T C A C C C A A A GC T G A G G A T A A T T C A C C CC T G A G G A T A A T T C A C C C A A A G C T T A
A A T T C A C C C A A A G C T T A G A A C AA C C C A A A G C T T A G A A C A
C T G A G G A T A A T T C A C C C A A A G C T T A G A A C AA G G A T A A T T C A C C C A A A G C T T A G A A C A
D Proband 3PIGG Exons 6-10Long Range PCR
6 87 9 10
chr4:516,000
500040003000200015001000
700500400300200
75
70001000020000
(bp)
518,000 520,000 522,000 524,000 526,000
Con
trol
Prob
and-
2
NG
S Pa
ired
Split
Rea
dsSa
nger
PIGG Exons
Intron 6
Intron 6
Intron 9
Intron 9
Targ
eted
NG
S
6.3 kb deletion (Exons 7-9)TGGCTAACACAGTGAGATTCTCCTGTCTCATGGCTAACACAGTGAGATTCTCCTGTCTCA
NGS Breakpoint Breakpoint Sequence
Contro
l
Φ Proba
nd 3
No DNA
Left
Rig
htA G T G A G A T T C T C C T G T C T C A
T G G C T A A C A C A G T G A G A T T C T C C T G T C T C A
A A C A C A G T G A G A T T C T C C T G T C T C AG A T T C T C C T G T C T C A
T G G C T A A C A C A G T G A G A T T C T C C T G T C T C AT G G C T A A C A C A G T G A G AT G G C T A A C A C A G T G A G A T T C T C CT G G C T A A C A C A G T G A G A T T C T C C T G T C T C A
paired split reads spanning deletion
NG_051621.1:g.5982_11944del
NG_051621.1:g.24216_30561del
DNARBC
WGS
Filtering
Emm− Proband 1All Variants
Proband 1Homozygous Variants
Homozygous inEmm− Brother
Heterozygous inEmm+ Children
gnomAD Allele Frequency <0.001Emm
Serologic Typing
Family Samples
BrotherEmm−
anti-Emm
Proband 1Emm−
anti-Emm
SpouseEmm+
DaugherEmm+
SonEmm+
A
B
C Variant IdentificationFamily Pedigree
Study Outline
5,149,248
1,942,938
1,427,285
425,464
1,818 1
Total Variants High Moderate Low
PIGG2 bp deletion, frameshift, premature stop
NM_001127178.3:c.2624_2625delTA
Variant Effect Predictor (VEP)
2 4
360 3,868 5,112
504 5,245 7,142
1,564 15,296 19,822
106 989 1,466
D WGS Alignments
G G C T - - G A CG G C T - - G A C
hg38:chr:533866_533874 (NM_001127178.3:c.2620_2628)
Proband 1
G G C T - - G A CG G C T - - G A C
G G C T T A G A CG G C T T A G A C
G G C T T A G A CG G C T - - G A C
G G C T T A G A CG G C T - - G A C
Brother Spouse Daughter SonEmm− Emm+
G G C T T A G A Chg38 ref
SampleSeq
G G C T T A G A C G G C T T A G A C G G C T T A G A C G G C T T A G A C
VEP ScoringAntibody ID
PIGG
A
Figure 4
Emm− Probands
B gnomAD Loss of Function
c.2624_2625delTAProband 1
c.1640G>AProband 5
Proband 3Proband 2
deletion Exons 2-3
Proband 4
Partial Deletion Exon 3
deletion Exons 7-9
frameshift (premature stop)
frameshift(premature stop)
new stop(reported LOF)
missense changeonly reported LOF
PIGG
c.2624_2625delTA
c.246dupTc.352C>T
c.624G>Ac.691delC
c.699_718dupGCAGAAGCTGAGCGAGATGG
c.1431T>Gc.1492_1493delTG
c.1515G>Ac.1562_1563delTGc.1562_1563delTG
c.2187_2200delGCTGGGCGTCTACT
c.2128delGc.2693G>A
c.928C>T
c.448delCc.473_474del
c.498G>Ac.768_769dupGA
c.801delT
c.1210delCc.1285C>T
c.1294delAc.1640G>A
c.1753delCc.1803delT
c.1898delG
c.1977G>Ac.2005C>T
c.1994delG
c.2029C>T
c.2391dupA
c.2569C>T
c.2778_2782delTTTGTc.2843G>A
c.2886delG
1 2 3 4 5 6 7 8 9 10 11 12 13
1 2 3 4 5 6 7 8 9 10 11 12 13
c.2444delC
c.2908_2909delGT
g.5982_11944del g.24216_30561del
c.361-51_383delinsGACTT
new stopin frame delins
P
3
2
1
P
3
2
1
P
3
2
1
P
3
2
1
P
2
1
P
2
1
P
1
P P
3
2
1
P
3
2
1
PGAP5PGAP1PIGKGPAA1PIGS
PIGT, PIGU
PIGFPIGG
PIGFPIGO
PIGBPIGNPIGV
H2H3 B
H6H7 H8
PIGMPIGX
6 7 8 9 10 11 12 13 14
Legend
Lumen
GlycanRemodeling
InositolDeacylationGPI Biosynthesis (PIG Genes)
ER
P
?
5P
PIGW
PIGAPIGCPIGHPIGPPIGQPIGYDPM2 PIGL 43
21
PPP ?
mix 1-alkyl-2-acyl & diacyl glycerol
diacyl glycerol
inositol phosphate glucosamine mannose
ethanolamine phosphate
(EtNP)linked protein
acylchain
Emm
P
Figure 5
N-acetylglucosamine
c.2624_2625delTA, p.(Leu875*), rs771819481
g.5982_11944del, p.(Ala53Glyfs*2)
g.24216_30561del, p.(Asp372Alafs*18)
Red cells from proband 5 and her brother, previously reported to have a PIGGloss of function variant [c.1640G>A, p.(Trp547*), rs547951371] identified by WES, were found here to be also be Emm− by serologic testing.
Proband 5 c.1640G>A, p.(Trp547*)
c.361-51_383delinsGACTT, p.(Ala121_Pro128delinsAspPhe)
Identification of PIGG Variants by targeted NGS and Sanger PIGG Variants Found in Emm− Probands 1-5
The five loss of function variants reported or investigated here are shown. Three causes a frameshift and premature stop codon, one causes a deletion and insertion, and one introduces a stop codon.
ConclusionThe findings here illustrate the power of using WGS with family cohorts to uncover the genetic basis of blood group systems. Based on these findings the ISBT Red Cell Immunogenetics and Blood Group Terminology working party has designated Emm as the 42nd blood group system.
The schematic shows the relevant part of the GPI anchor synthesis pathway. The proteins involved in each step are indicated below the line. PIGG transferase (bold) is reported to be expressed during erythropoiesis, but loss of function in Emm− individuals would mean EtNP is not added to mannose 2. EtNP on mannose 2 is the likely antigenic target responsible for anti-Emm.
Proposed Location of the Emm Antigen on GPI-anchors