Identification of novel, clinically correlated autoantigens in the monogenic autoimmune syndrome APS1 by proteome-wide PhIP-Seq
- Medical Scientist Training Program, University of California, San Francisco, United States
- Tetrad Graduate Program, University of California, San Francisco, United States
- Diabetes Center, University of California, San Francisco, United States
- Department of Biochemistry and Biophysics, University of California, San Francisco, United States
- Fungal Pathogenesis Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy & Infectious Diseases, National Institutes of Health, United States
- Biomedical Sciences Graduate Program, University of California, San Francisco, United States
- Department of Medicine, University of California, San Francisco, United States
- Department of Pediatrics, University of California, San Francisco, United States
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, United States
- Chan Zuckerberg Biohub, United States
Figures
Figure 1 with 2 supplements
PhIP-Seq identifies literature-reported autoantigens in APS1.
(A) Overview of PhIP-Seq experimental workflow. (B) PhIP-Seq identifies known autoantibody targets in APS1. Hierarchically clustered (Pearson) z-scored heatmap of literature reported autoantigens …
Figure 1—figure supplement 1
Hierarchically clustered (Pearson) z-scored heatmap of literature reported autoantigens that did not meet the cutoff of 10-fold or greater signal over mock-IP in at least 2/39 APS1 sera and in 0/28 non-APS1 control sera.
Figure 1—figure supplement 2
Additional PhIP-Seq data for known autoantigens SOX10 and NLRP5.
(A) Scatterplot of individual PhIP-Seq enrichment values (log10) over mock-IP as compared to radioligand binding assay antibody index values (1 = commercial antibody signal) for known antigens SOX10 …
Figure 2 with 1 supplement
PhIP-Seq identifies novel (and known) antigens across multiple APS1 sera.
(A) Hierarchically clustered (Pearson) z-scored heatmap of all genes with 10-fold or greater signal over mock-IP in at least 3/39 APS1 sera and in 0/28 non-APS1 sera. Black labeled antigens (n = 69) …
Figure 2—figure supplement 1
The mean of tissue-specificity ratio of 81 PhIP-Seq antigens (Figure 2) is increased as compared to the tissue-specificity ratio of n = 81 randomly sampled genes (n-sampling = 10’000).
Data from Protein Atlas, HPA/Gtex/Fantom5 RNA consensus dataset (https://www.proteinatlas.org/about/download; Uhlén et al., 2015).
Figure 3 with 1 supplement
Novel PhIP-Seq autoantigens are shared across multiple APS1 samples and validate in whole protein binding assays.
(A) Graph of the PhIP-seq autoantigens from Figure 2 that were shared across the highest number of individual APS1 sera (left panel). ASMT and PDX1 were positive hits in 3 and 2 sera, respectively, …
Figure 3—figure supplement 1
Comparison of PhIP-Seq data to orthonongal whole-protein binding assays.
(A) Scatterplot of individual PhIP-Seq enrichment values (log10) over mock-IP as compared to radioligand binding assay antibody index values (1 = commercial antibody signal) for novel antigens ACP4, …
Figure 4 with 2 supplements
PhIP-Seq reproduces known clinical associations with anti-CYP11A1 and anti-SOX10 antibodies.
(A) Heatmap of p-values (Kolmogorov-Smirnov testing) for differences in gene enrichments for individuals with versus without each clinical phenotype. Significant p-values in the negative direction …
Figure 4—figure supplement 1
Clustered disease correlations in the APS1 cohort (Spearman’s rank correlation; n = 67).
Figure 4—figure supplement 2
KHDC3L is highly expressed in oocytes (top), but not in granulosa cells (bottom).
In contrast, SRSF8 and PNO1 are highly expressed in granulosa cells (GCs), but not in oocytes. Data from Zhang et al. (2018).
Figure 5
Autoantibodies to oocyte-expressed protein KHDC3L are associated with ovarian insufficiency.
(A) Principle component analysis of transcriptome of single human oocytes (red) and granulosa cells (GCs, blue); data re-analyzed from Zhang et al. (2018). KHDC3L is highly expressed in oocytes, …
Figure 6 with 3 supplements
APS1 patients with intestinal dysfunction mount an antibody response to intestinal enteroendocrine cells and to enteroendocrine-expressed protein RFX6.
(A) Anti-RFX6 positive APS1 serum with intestinal dysfunction co-stains Chromogranin-A (ChgA) positive enteroendocrine cells in a nuclear pattern (right panel and inset). In contrast, non-APS1 …
Figure 6—figure supplement 1
Higher resolution PhIP-seq and transcriptional data for novel autoantigen RFX6.
(A) PhIP-Seq enables 49 amino acid resolution of antibody signal from novel autoantigen RFX6. PhIP-Seq signal (fold-change of each peptide as compared to signal from mock-IP, log10-scaled) for …
Figure 6—figure supplement 2
Anti-RFX6+ sera (top two panels), but not anti-RFX6- serum or non-APS1 control serum (bottom two panels), co-stain HEK293T cells transfected with an RFX6-expressing plasmid.
None of the sera tested stain 293T cells transfected with empty vector (‘mock’). No cross-reactivity of secondary antibodies was observed (right panel).
Figure 6—figure supplement 3
Extended validation and clinical correlations for intestinal antigens RFX6 and TPH1.
(A) APS1 patients with the diarrheal subtype, as well as those with both subtypes of ID (red), have increased anti-RFX6 antibody signal by RLBA as compared to those with constipation-type ID or no …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Transfected construct (human) | 293T mock transfection construct | Origene | PS100001 | |
| Transfected construct (human) | 293T RFX6 transfection construct | Origene | RC206174 | |
| Antibody | Goat Anti-NLRP5, polyclonal | Santa Cruz | sc-50630 | RLBA 1:50 |
| Antibody | Mouse Anti-SOX10, monoclonal | Abcam | ab181466 | RLBA 1:25 |
| Antibody | Sheep Anti-RFX6, polyclonal | R and D Systems | AF7780 | RLBA 1:50 |
| Antibody | Rabbit Anti-KHDC3L, polyclonal | Abcam | ab170298 | RLBA 1:25 |
| Antibody | Rabbit Anti-CYP11A1, polyclonal | Abcam | ab175408 | RLBA 1:50 |
| Antibody | Rabbit Anti-NKX6-3, polyclonal | Biorbyt | orb127108 | RLBA 1:50 |
| Antibody | Mouse Anti-GIP, monoclonal | Abcam | ab30679 | RLBA 1:50 |
| Antibody | Rabbit Anti-PDX1, polyclonal | Invitrogen | PA5-78024 | RLBA 1:50 |
| Antibody | Rabbit Anti-ASMT, polyclonal | Invitrogen | PA5-24721 | RLBA 1:25 |
| Antibody | Rabbit Anti-CHGA, polyclonal | Abcam | ab30679 | IF 1:5000 |
| Antibody | Rabbit Anti-DYKDDDDK (D6W5B), monoclonal | Cell Signaling Technologies | #14793 | RLBA 1:125, IF 1:2000 |
Additional files
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Supplementary file 1
APS1 cohort: Clinical Data.
ND, nail dystrophy. HP, hypoparathyroidism. KC, keratoconjunctivitis. CMC, chronic mucocutaneous candidiasis. ID (D, C, B), Intestinal dysfunction (diarrheal-type, constipation-type, both). AIH, autoimmune hepatitis. POI, primary ovarian insufficiency. HTN, hypertension. HT, hypothyroidism. B12 def, B12 (vitamin) deficiency. DM, diabetes mellitus. SS, Sjogren’s-like syndrome. GH def, Growth hormone deficiency. AI, Adrenal Insufficiency. EH, (dental) enamel hypoplasia. TF, testicular failure. TIN, Tubulointerstitial Nephritis. Hpit, Hypopituitarism. UE, Urticarial eruption. D, Discovery cohort; V, Validation cohort. *Age at most recent evaluation
- https://cdn.elifesciences.org/articles/55053/elife-55053-supp1-v1.docx
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Supplementary file 2
Non-APS1 control cohort: Clinical Data.
D, Discovery cohort; V, Validation cohort.
- https://cdn.elifesciences.org/articles/55053/elife-55053-supp2-v1.docx
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Supplementary file 3
Tissue-restricted expression patterns of validated and putative novel APS1 antigens.
- https://cdn.elifesciences.org/articles/55053/elife-55053-supp3-v1.docx
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Supplementary file 4
Antibody information by application.
- https://cdn.elifesciences.org/articles/55053/elife-55053-supp4-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/55053/elife-55053-transrepform-v1.docx
