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