Uncovering potential single nucleotide polymorphisms, copy number variations and related signaling pathways in primary Sjogren’s syndrome

Figures & data

Table 1. Clinical information of enrolled 12 individuals in the WES analysis

Patient	Age	Clinical features	Complications	Family history	Ro/La antibodies	Levels of immunoglobulin G (g/L)
AII2	50	Dry mouth, dry eyes (for 10 years), hyperglobulinemia, mumps and peripheral neuropathy	Thyroid nodules and peripheral neuropathy	Yes	Positive for SSA and SSB	29.1
AI2	70	Teeth flaky loss for 10 years	None	Yes	Positive for SSA and SSB	19
AIII1	28	None	None	Yes
BI2	58	Dry mouth and dry eyes (for 2 years)	Hyperthyroidism	Yes	Positive for SSA and SSB	15.2
BII2	30	None	None	Yes
CI2	36	Both lower limbs purpura for 7 years and hyperglobulinemia	None	No	Positive for SSA and SSB	22.4
CII1	9	None	None	No
D1	75	Dry mouth and flaky tooth loss (over 20 years)	Pulmonary interstitial fibrosis with infection	No	Positive for SSA	14.1
D2	49	Teeth flaky loss (for 10 years) and dry mouth and dry eyes (for 5 years)	Liver cirrhosis	No	Positive for SSA and SSB	33.8
D3	31	Dry mouth (for 2 years) and the whole body fatigue (for 0.5 year)	Renal tubular acidosis	No	Positive for SSA and SSB	36.3
D4	70	Teeth flake loss (for 8 years) and dry mouth	Thrombocytopenia	No	Positive for SSA and SSB	16.4
D5	63	Repeated intrahepatic calculi for 14 years	Intrahepatic bile duct stones	No	Negative for SSA and SSB	26

A, B, and C represent individual in the pedigrees; D represents individual in the sporadic cases.

Figure 1. Three pSS pedigrees examined in this study

The box and circle represent male and female, respectively. The black color represents individual with pSS. A, B, and Crepresent pedigrees 1, 2, and 3, respectively. W: individual that enrolled for WES analysis.

Table 2. Common mutation genes between pedigree A, B, and C

Gene	Start_end	Func.refGene	AI2	AII2	BI2	CI2
SSU72	1541728_1541728	UTR3	G→A	G→A	G→A	G→A
SSU72	1541943_1541945	UTR3	GCA→G	GCA→G	GCA→G	GCA→G
LOC105370401	22483099_22483099	Upstream	A→G	A→G	A→G	A→G
ELP4\PAX6	31789577_31789577	UTR3	T→TA	T→TA	T→TA	T→TA
HLA-DRB1	32578815_32578815	UTR3	A→G	A→G	A→G	A→G
TAF1L	32635707_32635708	Upstream	CT→C	CT→C	CT→C	/
ZNF180	44477666_44477666	Exonic	G→C	G→C	G→C	G→C
ZNF180	44479350_44479350	Exonic	G→C	G→C	G→C	G→C
C18orf25	46253735_46253738	Exonic	TCTG→T	TCTG→T	TCTG→T	TCTG→T
KCNB1	49483100_49483100	Upstream	C→CT	/		/
DDX28	68021475_68021475	UTR3	C→T	C→T	C→T	C→T
FOXC2	86569037_86569037	UTR3	C→T	C→T	C→T	C→T
CWF19 L1	100233166_100233167	UTR3	TA→T	TA→T	TA→T	TA→T
BTLA	112499472_112499472	UTR5	T→TAA	/	/	/
LOC653513	149197754_149197754	ncRNA_exonic	T→C	T→C	T→C	T→C
LOC285097	242003892_242003892	ncRNA_exonic	C→T	C→T	C→T	C→T

Figure 2. Number and functional analysis of mutant genes in pedigree A

A: Venn diagrams of mutations between AI2, AII2, and AII1 individuals. B: top 10 significantly enriched biological processes of mutant genes. C: all significantly enriched signal pathways of mutant genes.

Figure 3. Number and functional analysis of mutant genes in pedigree B

A: Venn diagrams of mutations between BI2 and BII2 individuals. B: top 10 significantly enriched biological processes of mutant genes. C: all significantly enriched signal pathways of mutant genes.

Figure 4. Number and functional analysis of mutant genes in pedigree C

A: Venn diagrams of mutations between CI2 and CII1 individuals. B: all significantly enriched GO terms of mutant genes. C: all significantly enriched signal pathways of mutant genes.

Table 3. Mutation information of FCGBP, ANKRD36C, and FRG2C in the sporadic cases

Gene	Start_end	Func.refGene	D1	D2	D3	D4	D5
FCGBP	39886243_39886243	Exonic	C→G	C→G	C→G	C→G	C→G
FCGBP	39886257_39886257	Exonic	C→T	C→T	C→T	C→T	C→T
FRG2C	75665673_75665674	Exonic	AG→A	AG→A	AG→A	AG→A	AG→A
FRG2C	75665948_75665948	Exonic	T→A	T→A	T→A	T→A	T→A
ANKRD36C	95950756_95950756	Exonic	C→A	C→A	C→A	C→A	C→A
ANKRD36C	95950758_95950758	Exonic	G→C	G→C	G→C	G→C	G→C

Figure 5. Common mutations and PPI analysis of mutant genes in 5 sporadic cases

A: Venn diagrams of common mutations. B: Reactome analysis of common mutant genes. C: PPI analysis of common mutant genes.

Table 4. Common mutation genes in family patients and sporadic patients

Gene	Start	Position	AI2	AII2	AIII3	BI2	BII2	CI2	CII1	D1	D2	D3	D4	D5
FRG1DP\FRG2EP	29327738	Intergenic	G→A	G→A	/	G→A	/	G→A	/	G→A	G→A	G→A	G→A	G→A
FES	90891941	Intronic	G→C	G→C	/	G→C	/	G→C	/	G→C	G→C	G→C	G→C	G→C
PPM1J	112713064	Intronic	T→TT	T→TT	/	/	/	/	/	/	/	/	T→TT	/
TRAPPC9	139,971,349	Intronic	C→T	C→T	/	C→T	/	C→T	/	C→T	C→T	C→T	C→T	C→T

Table 5. Partial pathogenic genes in the CNV deletion regions and involved systemic lupus erythematosus signaling pathway in family A

Chr	Start_end	Length	Partial genes	CNV status
6	24865529_26370831	1,505,302	HIST1H4A:F|TRIM38:F|HIST1H4G:F|HIST1H4F:F|HIST1H4C:F|HIST1H4B:F|HIST1H4H:F|LRRC16A:F|SLC17A1:F|HIST1H1D|RP11-191A15.1:F|RP11-191A15.2:F|HIST1H2BG:F|RP11-191A15.4:F|HIST1H3D:F|HIST1H3F:F|HIST1H2BE:F|U91328.22:F|U91328.21:F|U91328.20:F|RNY5P5:F	Deletion
6	31515940_31659457	143,517	GPANK1:F|BAG6:F|LY6G5C:F|LST1:F|PRRC2A:F|APOM:F|NCR3:F|UQCRHP1:F|C6orf47:F|LY6G5B:F|LTB:F|LTA:F|TNF:F|C6orf47-AS1:F|CSNK2B:F|CSNK2B-LY6G5B-1181:F| AIF1:F	Deletion
2	170462549_171258201	795,652	KLHL23:F|AC016772.4:F|PTCHD3P2:F|CCDC173:F|METTL5:F|AC012594.1:F|PPIG:P|UBR3:F|PHOSPHO2:F|RNU6-1006P:F|SSB:F	Deletion

Table 6. Partial pathogenic genes in the CNV deletion regions in family B

Chr	Start_end	Length	Partial genes	CNV status
2	170462549_171258201	795,652	KLHL23:F|AC016772.4:F|PTCHD3P2:F|CCDC173:F|METTL5:F|AC012594.1:F|PPIG:P|UBR3:F|PHOSPHO2:F|RNU6-1006P:F|SSB:F	Deletion
2	31515940_31659457	143,517	GPANK1:F|BAG6:F|LY6G5C:F|LST1:F|PRRC2A:F|APOM:F|NCR3:F|UQCRHP1:F|C6orf47:F|LY6G5B:F|LTB:F|LTA:F|TNF:F|C6orf47-AS1:F|CSNK2B:F|CSNK2B-LY6G5B-1181:F| AIF1:F	Deletion

Figure 6. Pathogenic CNVs and functional analysis of involving genes in pedigree A

A: Venn diagrams of pathogenic CNVs between AI2, AII2, and AII1 individuals. B: top 10 significantly enriched biological processes of involving genes in CNVs regions. C: all significantly enriched signal pathways of involving genes in CNVs regions.

Figure 7. Pathogenic CNVs and functional analysis of involving genes in pedigree B

A: Venn diagrams of pathogenic CNVs between BI2 and BII2 individuals. B: top 10 significantly enriched biological processes of involving genes in CNVs regions. C: all significantly enriched signal pathways of involving genes in CNVs regions.

Figure 8. Pathogenic CNVs and functional analysis of involving genes in pedigree C

A: Venn diagrams of pathogenic CNVs between CI2 and CII1 individuals. B: top 10 significantly enriched biological processes of involving genes in CNVs regions.C: all significantly enriched signal pathways of involving genes in CNVs regions.

Figure 9. Venn diagrams of pathogenic CNVs in 5 sporadic cases

Table 7. 3 GEO datasets of gene expression in pSS

Dataset	Sample	Microarray analysis	Group
GSE66795	Whole blood	Illumina	Control(29)+pSS(131)
GSE145065	Peripheral blood monouclear cell	Illumina	Control(5)+pSS(5)
GSE84844	Whole blood	Affymetrix	Control(30)+pSS(30)

Table 8. CNVs amplification/deletion of 51 immune related genes in 3 pedigrees

Sample	Chr	Start_end	Length	Partial genes	CNV status
Pedigree A	1	211960974_213139120	1,178,146	BATF3:F|PPP2R5A:F|ATF3:F	Deletion
Pedigree B	3	122044140_122662382	618,242	DTX3L:F|PARP9:F|PARP15:F|HSPBAP1:F|EIF4BP8:F|CCDC58:F	Duplication
Pedigree B	10	5960305_6002530	42,225	FBXO18:P|IL15RA:P|RP11-536K7.3:F	Deletion
Pedigree B	10	6005706_6258743	253,037	IL2RA:F|RP11-414 H17.5:F|RP11-414H17.2:F|PFKFB3:P|RP11-536K7.5:F|RBM17:F|IL15RA:P	Deletion
Pedigree B	3	122121607_122459960	338,353	DTX3L:F|HSPBAP1:P|PARP9:F	Deletion
Pedigree C	11	64502584_64889285	386,701	SYVN1:P|CDC42BPG:F|CDCA5:F|AP000436.4:F|PYGM:F|ZNHIT2:F|BATF2:F|VPS51:F|MIR194-2:F|PPP2R5B:F	Deletion
Pedigree C	7	92252350_92821676	569,326	AC002454.1:F|SAMD9L:F|HEPACAM2:P|CDK6:P|RN7SL7P:F|SAMD9:F	Duplication

Table 9. CNVs amplification/deletion of 51 immune related genes in 5 sporadic cases

Sample	Chr	Start_end	Length	Partial genes	CNV status
D1	1	155721751_155932974	211,223	KIAA0907:F|ARHGEF2:P|RXFP4:F|RIT1:F|RNU4-19P:F|SYT11:F|GON4L:P|RP11-101O6.2:F	Duplication
D1	1	160616660_160769872	153,212	CD48:F|SETP9:F|LY9:P|RP11-404 F10.2:F|SLAMF7:F|SLAMF1:P	Duplication
D2	10	5959553_6008302	48,749	FBXO18:P|IL15RA:P|RP11-536K7.3:F	Deletion
D2	10	6008108_6258743	250,635	IL2RA:F|RP11-414H17.5:F|RP11-414H17.2:F|PFKFB3:P|RP11-536K7.5:F|RBM17:F|IL15RA:P	Deletion
D2	10	7327828_7622027	294,199	RNU6-535P:F|SFMBT2:P|RP5-1092K5.2:F|RP11-385N23.1:F|ITIH5:P|RP5-1031D4.2:F	Deletion
D2	11	64502584_64889285	386,701	CDCA5:F|AP000436.4:F|PYGM:F|ZNHIT2:F|BATF2:F	Deletion
D3	3	122121607_124721159	2,599,552	PARP9:F|ROPN1:F|PDIA5:F|DIRC2:F|MUC13:F|RP11-521 J5.1:F|SEMA5B:F|ADCY5:F|SEC22A:F|HSPBAP1:F|FAM162A:P|PARP14:F|	Deletion
D3	4	17510894_17830011	319,117	MED28:F|snoU13:F|QDPR:P|LAP3:F|AC006160.5:F|AC006160.4:F|NCAPG:P|CLRN2:F|FAM184B:F|DCAF16:F	Deletion
D3	8	90801549_90976735	175,186	OSGIN2:F|COX6B1P6:F|RIPK2:P|RNU6-925P:F|NBN:P	Deletion
D3	11	57317441_57461387	143,946	YPEL4:F|ZDHHC5:P|CLP1:F|UBE2L6:F|AP000662.4:F|SERPING1:F|RPS4XP13:F|SMTNL1:P	Deletion
D3	11	64502584_64889285	386,701	BATF2:F|VPS51:F|MIR194-2:F|PPP2R5B:F	Deletion
D4	3	122121607_122459960	338,353	PARP9:F|RP11-592P9.1:F|RP11-299J3.6:F|PARP15:F|WDR5B:F|KPNA1:F	Deletion
D4	10	5960305_6258743	298,438	IL2RA:F|FBXO18:P|RP11-414H17.2:F|PFKFB3:P|RP11-414H17.5:F|RP11-536K7.5:F|RBM17:F|RP11-536K7.3:F|IL15RA:F	Deletion
D4	11	64502584_64889285	386,701	|BATF2:F|VPS51:F|MIR194-2:F|PPP2R5B:F	Deletion
D4	12	113346341_113554966	208,625	DTX1:F|RP1-71H24.1:P|RP1-71H24.4:F|OAS1:P|OAS3:F|OAS2:F|RASAL1:P|RPS15AP32:F	Duplication
D5	3	122121607_122459960	338,353	PARP9:F|RP11-592P9.1:F|RP11-299J3.6:F|PARP15:F|WDR5B:F|KPNA1:F|EIF4BP8:F|RP11-299J3.8:F|PARP14:F|FAM162A:P	Deletion

Figure 10. GSEA enrichment pathways of differentially expressed genes in both GSE66795 dataset and GSE84844 dataset

A, C, and E respectively represent IL2-STAT5 signaling, interferon-gamma response and interferon-alpha response in GSE66795 dataset. B, D, and F respectively represent IL2-STAT5 signaling, interferon-gamma response and interferon-alpha response in GSE84844 dataset.

Figure 11. Sanger validation results of ZNF180 variant in 4 patients in 3 pedigrees and 2 sporadic patients. Red base represents the mutation site

Figure 12. Sanger validation results of FCGBP variant in 4 patients in 3 pedigrees and 5 sporadic patients. Red base represents the mutation site

Figure 13. Sanger validation results of FRG2C variant in 4 patients in 3 pedigrees and 5 sporadic patients. Red base represents the mutation site

Figure 14. The in vitro validation of SSB, BATF2, BATF3, PARP9, IL15RA, and LAP3 in pSS

Data availability statement

All data are available in the article.