A Deep Dive into the Pathology of Gray Platelet Syndrome: New Insights on Immune Dysregulation

Figures & data

Table 1 Published NBEAL2 Variants in GPS Patients

Pedigrees	Mutation state	Genotype	Predicted protein effect	Type of mutation	Ref.
1	homozygous	c.2701C>T	p.R901*	nonsense	^Citation7,Citation16
2	homozygous	c.881C>G	p.S294*	nonsense	^Citation7,Citation16
3	homozygous	c.1163T>C	p.L388P	missense	^Citation7,Citation16
4	homozygous	c.5720+5G>A	NA	splicing	^Citation7,Citation16
5	homozygous	c.5515C>T	p.R1839C	missense	^Citation7,Citation16
6	homozygous	c.1296+5G>C	NA	splicing	^Citation7,Citation16
7	homozygous	c.2257_2260delGCCC	p.A753Sfs*65	frameshift	^Citation7,Citation16
8	homozygous	c.1296+5G>C	NA	splicing	^Citation7,Citation16
9	homozygous	c.3819_4174del356	p.V1274Gfs*32	frameshift	^Citation7,Citation16
10	homozygous	c.2029T>A	W677R	missense	^Citation7,Citation16
11	homozygous	c.7604delG	p.G2535Vfs*5	frameshift	^Citation7,Citation16
12	homozygous	c.5505T>G	p.Y1835*	nonsense	^Citation7,Citation16
13	compund heterozygous	c.2701C>T	p.R901*	nonsense	^Citation7,Citation16
		c.6787C>T	p.H2263Y	missense
14	compund heterozygous	c.2156delT	p.F719Sfs*100	frameshift	^Citation7,Citation16
		c.5497G>A	p.E1833K	missense
15	homozygous	c.5301+1G>A	NA	splicing	^Citation7,Citation16
16	compund heterozygous	c.1029+1G>A	NA	splicing	^Citation5,Citation16
		c.4371_4375dupCGTGG	p.E1459Afs*43	frameshift
17	homozygous	c.1820G>A	p.W607*	nonsense	^Citation5,Citation16
18	compund heterozygous	c.5413dupG	p.A1805Gfs*59	frameshift	^Citation5,Citation16
		c.1820G>A	p.W607*	nonsense
19	homozygous	c.1163T>C	L388P	missense	^Citation6,Citation8,Citation16
20	homozygous	c.2044A>T	I682F	missense	^Citation6,Citation8,Citation16
21	compound heterozygous	c.1928A>T	E643V	missense	^Citation6,Citation8,Citation16,Citation18
		c.6299C>T	P2100L	missense
22	compound heterozygous	c.1504_1507del	p.L502Afs*4	frameshift	^Citation10,Citation16
		c.6801+7A>T	p.E2268Vfs*44	splicing
23	compound heterozygous	c.5572C>T	p.R1858*	nonsense	^Citation8,Citation10,Citation16
		c.6652G>T	p.E2218*	nonsense
		c.7033C>T	p.R2345W	missense
24	homozygous	c.2187C>A	p.Y729*	nonsense	^Citation10,Citation16
25	compund heterozygous	c.1253del	p.H418Lfs*54	frameshift	^Citation8,Citation10,Citation16
		c.3584G>A	p.R1195Q	missense
		c.5720+1G>A	p.M1908*	splicing
26	homozygous	c.5299C>T	p.Q1767*	nonsense	^Citation11,Citation16,Citation18
27	homozygous	c.del4501-4503	p.L1501del	deletion	^Citation11,Citation16
28	homozygous^#	c.881C>G	p.S294*	nonsense	^Citation11,Citation16
		c.958C>T	p.R320W	missense
29	homozygous	c.7440G>A	p.W2480*	nonsense	^Citation8,Citation12,Citation16
30	homozygous	c.5721-1G>C	p.M1908*	splicing	^Citation8,Citation12,Citation16
31	homozygous	c.6212G>C	p.R2071P	missense	^Citation13,Citation16
32	compound heterozygous	c.3839C>T	p.R1280*	nonsense	^Citation13,Citation16
		c.6477C>G	p.H2159Q	missense
33	homozygous	c.5176G>T	p.E1726*	nonsense	^Citation14,Citation16
34	homozygous	c.7225-1G>C	NA	splicing	^Citation8,Citation15
35	compund heterozygous	c.1870C>T	p.R624*	nonsense	^Citation16
		c.2735dupG	p.P913Sfs*3	frameshift
36	homozygous	c.352-1_352delinsTA	NA	splicing	^Citation16
37	compund heterozygous	c.3384 + 5G>A	NA	splicing	^Citation17
		c.5965G>A	p.E1989K	missense
38	homozygous	c.7387C>T	p.Q2463*	nonsense	^Citation8
	compund heterozygous^ϕ	c.6657C>A	p.F2219L	missense
39	compound heterozygous	c.4081G>T	p.E1361*	nonsense	^Citation8
		c.1793G>A	p.W598*	nonsense
40	compund heterozygous	c.3118+2T>G	NA	splicing	^Citation8
		c.6959G>C	p.R2320P	missense
41	homozygous	c.6432delT	p.F2144Lfs*23	frameshift	^Citation8
42	homozygous	c.1725_1728dupACGT	p.A577Tfs*7	frameshift	^Citation8
43	homozygous	c.3773_3780delinsTCAGCGTTCGCCTCAGA	p.N1259_I1260delinsSVRLR	indel	^Citation8
44	homozygous	c.6460T>C	p.F2154L	missense	^Citation8
45	homozygous	c.4928_4929delAT	p.D1643Gfs*34	frameshift	^Citation8
46	homozygous	c.4081G>T	p.E1361*	nonsense	^Citation8
47	homozygous	c.881C>G	p.S294*	nonsense	^Citation8
48	homozygous	c.607dupA	p.I203Nfs*21	frameshift	^Citation8
49	compound heterozygous	c.1163T>C	p.L388P	missense	^Citation8
		c.6202T>C	p.W2068R	missense
50	homozygous	c.7192_7202dupTCCTTCATCAC	p.Q2402Pfs*12	frameshift	^Citation8
51	homozygous	c.6359G>A	p.R2120Q	missense	^Citation8
52	homozygous	c.4890delG	p.R1631Gfs*3	frameshift	^Citation8,Citation18
53	homozygous	c.7506delT	p.D2503Mfs*2	frameshift	^Citation8
54	compund heterozygous	c.6568delT	p.C2190Afs*23	frameshift	^Citation8,Citation18
		c.7937T>C	p.L2646P	missense
55	compound heterozygous	c.2701C>T	p.R901*	nonsense	^Citation8
		c.2552C>T	p.P851L	missense
56	homozygous	c.6515G>A	p.R2172H	missense	^Citation8
57	compund heterozygous	c.5935C>T	p.R1979W	missense	^Citation8
		c.6868G>T	p.G2290W	missense
		c.6881A>G	p.E2294G	missense
58	compound heterozygous	c.2650-1G>A	NA	splicing	^Citation8,Citation18
		c.3058T>C	p.Y1020H	missense
		c.7134G>A	NA	splicing
59	compound heterozygous	c.6787C>T	p.H2263Y	missense	^Citation8
		c.6894T>A	p.N2298K	missense
60	compound heterozygous	c.1789C>T	p.R597*	nonsense	^Citation8
		c.6359G>A	p.R2120Q	missense
61	homozygous	c.6239T>A	p.M2080K	missense	^Citation8
62	homozygous	c.1476_1479dupAGGC	p.L494Rfs*59	frameshift	^Citation8
63	homozygous	c.2537T>C	p.L846P	missense	^Citation8
64	homozygous	c.7501C>T	p.H2501Y	missense	^Citation8
65	compound heterozygous	c.427G>A	p.E143K	missense	^Citation8
		c.2751dupT	p.D918*	nonsense
66	compound heterozygous	c.256A>G	p.I86V	missense	^Citation6,Citation8,Citation18
		c.6805_6806insAGGGAGT	p.S2269*	splicing
		c.6920-1G>C	NA	splicing
67	compound heterozygous	c.4485-1G>T	NA	splicing	^Citation8,Citation18
		c.1936_1938delTTC	p.F646del	deletion
68	compound heterozygous	c.6432delT	p.F2144Lfs*23	frameshift	^Citation18
		c.5497G>A	p.E1833K	missense
69	homozygous	c.5674C>T	p.Q1829*	nonsense	^Citation19

Notes: NBEAL2 variants are named according to reference sequence NM_015175. Intronic variants located at splice junctions, predicted to affect splicing, are listed as splicing variants. Mutations detected in more than one pedigree are depicted in bold. All references (Ref.) where the variants are described are included. ^# both variants (p.S294* and p.R320W) were found in homozygosis in pedigree 28. ^ϕ in addition to the homozygous variant (p.Q2463*) found in the two affected individuals from pedigree 38, one of them harboured an additional heterozygous variant (p.F2219L).

Table 2 Abnormalities in Innate and Acquired Immunity in GPS Patients and Animal Models

	Human GPS	Nbeal2-null mice
Neutrophil granule content
All granules	Normal^Citation3,Citation44 (EM)	Reduced^Citation47 (EM)
Azurophilic granules	Normal^Citation25,Citation43 (IEM)	Reduced^Citation48 (EM)
Specific granules	Reduced^Citation8,Citation25,Citation43 (IEM)	Reduced^Citation48 (EM)
Gelatinase granules	Reduced^Citation8 (MS)	Reduced^Citation48 (EM)
Secretory vesicles	Reduced^Citation25 (Cyto)	Reduced^Citation48 (EM)
Surface expression of granule proteins
CD11b (SG and SV)	Increased^Citation25,Citation43	Increased^Citation48
CD66b (SG)	Increased^Citation18	NE
CD35 (SV)	Increased^Citation25,Citation43	NE
Release of granule content
Elastase (AG)	Normal^Citation18	Decreased^Citation47
Lactoferrin (SG)	Decreased^Citation18	NE
Proteomic analysis
SG and GG content in neutrophils	Downregulated^Citation8,Citation18	Downregulated*^Citation47
Neutrophil proteins in plasma	Upregulated^Citation8	Upregulated**^Citation48
Neutrophil proteins in platelets	Upregulated^Citation18	NE
Neutrophil functions
Reactive oxygen species production	Normal^Citation18,Citation25	Enhanced^Citation47, Normal^Citation48
Bacterial killing or phagocytosis	Normal^Citation18	Normal^Citation48
Migration	Normal^Citation25	Enhanced^Citation48
NETosis	Impaired^Citation18	Normal^Citation48
Suceptibility to infection	Increased^Citation8	Increased^Citation47,Citation48
NK cells
NK numbers	NE	Reduced^Citation47
NK degranulation	NE	Impaired^Citation47
Autoimmunity
Autoantibodies	Increased^Citation8	NE
Autoimmune disease	Increased^Citation8	NE
Immune response markers in CD4+ cells	Elevated^Citation8	NE
Inflammation
Acute-phase reactants	Elevated^Citation8	NE

Notes: Techniques used to assess neutrophil granule content are detailed in parenthesis. *AG content was also shown to be downregulated. **as determined by ELISA.

Abbreviations: EM, electron microscopy; IEM, immune electron microscopy; MS, mass spectrometry; Cyto, cytochemistry; SG, specific granules; SV, secretory vesicles; AG, azurophilic granules; NE, not evaluated.

Figure 1 NBEAL2 variants in GPS patients. Schema of the NBEAL2 protein comprising the ARM (armadillo)-like domain, spanning amino acids (aa) 377–504, the Con (concanavalin) A-like lectin domain (aa 580–730), the PH (pleckstrin homology) domain (aa 1915–2040), the BEACH (beige and Chediak-Higashi syndrome domain (aa 2053–2345) and WD40 repeat domains (2463–2722), according to Sims et al.⁸ Published missense, nonsense, frameshift, indel and splicing germline NBEAL2 variants described in GPS pedigrees (Table 1) are indicated by the symbols. Missense variants are enriched within the BEACH domain. The position of splicing variants is given according to the predicted effect at the protein level.

Figure 2 Pale platelets on the blood smear from a GPS patient. Large pale platelets due to absence or marked decrease in α-granules is a typical finding in GPS patients. A May-Grünwald Giemsa-stained blood smear from a GPS patient is shown on the right panel. Gray platelets are indicated by the arrows. A blood smear from a healthy subject stained simultaneously is shown on the left panel. Images were obtained at 1000x magnification.

Figure 3 Abnormalities in the innate and adaptive immune response in GPS. GPS neutrophils display reduced numbers of specific (red circles) and gelatinase (yellow circles) granules. Whereas azurophilic (blue circles) granules are preserved in GPS patients, they are decreased in GPS animal models. Proteins resident in specific and gelatinase granules are expressed at the cell membrane, such as CD11b, CD66b and CD35, and elevated in plasma, indicating degranulation. Neutrophil extracellular trap formation (NETosis) is impaired. NK cell number and function are decreased in GPS animal models, although it is unknown whether it is also altered in patients. Altogether, these abnormalities may contribute to higher susceptibility to infections. Autoimmune manifestations and/or autoantibodies (AutoAb) are present in around half of GPS patients, coupled to upregulation of immune response markers in CD4+ cells. Liver-derived acute phase reactants are elevated in patient circulation, reflecting ongoing inflammation.

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