Diabetes Page 2 of 61
Carboxyl-ester lipase Maturity-Onset Diabetes of the
Young is Associated with Development of Pancreatic
Cysts and Upregulated MAPK Signaling in Secretin-
stimulated duodenal fluid
Helge Ræder 1,2,3, Fiona E. McAllister4*, Erling Tjora2,3*, Shweta Bhatt1, Ingfrid
Haldorsen5,6, Jiang Hu1, Stefan M. Willems4, Mette Vesterhus7, Abdelfattah El Ouaamari1,
Manway Liu8, Maria B. Raeder3, Heike Immervoll9,10,11, Dag Hoem12, Georg Dimcevski7 ,
Pål R. Njolstad2,3, Anders Molven9,10, Steven P Gygi4, Rohit N. Kulkarni1
From the 1Section of Islet Cell Biology and Regenerative Medicine, Joslin Diabetes Center,
Harvard Medical School, Boston, MA, USA, 2Department of Pediatrics, Haukeland University
Hospital, Bergen, Norway; 3Department of Clinical Science, University of Bergen, Bergen,
Norway, 4Department of Cell Biology, Harvard Medical School, Boston, MA, USA,5Department
of Radiology, Haukeland University Hospital, Bergen, Norway; 6Section for Radiology,
Department of Clinical Medicine, University of Bergen, 7Department of Medicine, Haukeland
University Hospital, Bergen, Norway, 8Department of Biomedical Engineering, Boston
University, Boston, MA,USA , 9The Gade Laboratory for Pathology, Department of Clinical
Medicine, University of Bergen, Norway; 10Department of Pathology, Haukeland University
Hospital, Bergen, Norway, 11Department of Pathology, Ålesund Hospital, Ålesund, Norway;
12Department of Surgery, Haukeland University Hospital, Bergen, Norway, *,These authors
contributed equally.
Corresponding author: Helge Raeder, [email protected]
1
Diabetes Publish Ahead of Print, published online September 23, 2013 Page 3 of 61 Diabetes
Running title: PANCREATIC CYSTS AND MAPK SIGNALING IN CEL-MODY
Original article: Word count, abstract: 200, Word count, main text: 3161, Number of references:
33, Number of figures: 5, Number of tables: 2.
ABSTRACT
CEL MODY is a monogenic form of diabetes and pancreatic exocrine dysfunction due to
mutations in the carboxyl ester lipase gene CEL. The pathogenic mechanism for diabetes
development is unknown. Since CEL is expressed mainly in pancreatic acinar cells, we asked
whether we could find structural pancreatic changes in CEL MODY subjects during the course
of diabetes development. Furthermore, we hypotesized that the diseased pancreas releases
proteins which are detectable in pancreatic fluid and potentially reflected activation or
inactivation of disease specific pathways. We therefore investigated non diabetic and diabetic
CEL mutation carriers by pancreatic imaging studies and secretin stimulated duodenal juice
sampling. The secretin stimulated duodenal juice was studied using cytokine assays, triple stage
mass spectrometry (MS3) and multiplexed mass spectrometry based measurement of kinase
activities. We identified multiple pancreatic cysts in all the eight diabetic mutation carriers but
not in any of the four non diabetic mutation carriers or the six healthy controls. Furthermore, we
identified upregulated MAPK target proteins and MAPK driven cytokines and increased MAPK
activity in the secretin stimulated duodenal juice. These findings show that subjects with CEL
MODY develop multiple pancreatic cysts by the time they develop diabetes and that upregulated
MAPK signaling in the pancreatic secretome may reflect the pathophysiological development of
pancreatic cysts and diabetes.
Keywords: MODY; Carboxyl ester lipase, pancreatic cysts, proteome profiling; MAPK signaling
2
Diabetes Page 4 of 61
INTRODUCTION
Studies of patients with Maturity onset Diabetes of the Young have provided important insight into disease mechanisms of pancreatic beta cell failure and diabetes development and also contributed to novel treatment strategies based on this insight (1; 2). We have previously reported two families with a human monogenic syndrome of diabetes and pancreatic exocrine dysfunction
(maturity onset diabetes of the young, type 8, MODY8; CEL MODY;OMIM #609812) caused by mutations in the carboxyl-ester lipase (CEL) gene (3). Patients with CEL mutations develop pancreatic exocrine dysfunction in early childhood (as measured by low fecal elastase levels), accumulate pancreatic fatty tissue, and develop diabetes and clinical malabsorption in their fourth decade of life (3; 4). The CEL gene is primarily expressed in pancreatic acinar cells and lactating mammary tissue, and encodes a digestive enzyme with a role in cholesterol ester digestion (5).
Studies of animal models have not been able to explain the disease mechanism of diabetes development in CEL MODY (6; 7), but cellular studies indicate that mutated CEL protein is misfolded (8). Hence, to gain further insight into the disease mechanism, we further studied human CEL mutation carriers using radiological methods and secretin stimulated duodenal juice to examine the pancreatic proteome (9). We hypotesized that the diseased pancreas releases proteins which are detectable in pancreatic fluid and potentially reflected activation or inactivation of disease specific pathways. To optimize the accuracy of the proteomics studies we applied triple stage mass spectrometry, MS3 (10), and complemented this approach with cytokine assays and MS based multiplex kinase activity assays (11) to cross validate our findings.
3
Page 5 of 61 Diabetes
RESEARCH DESIGN AND METHODS
Patients. We have previously reported various clinical characteristics for the CEL mutation
carrying subjects (3). In this study the studied CEL mutation carrying subjects (CEL+) were
denoted with the prefix D if they had manifest diabetes (D1, D2, D3 and D4), with the prefix P if
they had not yet developed diabetes (P1 or P2), or with the prefix C for the patient with
pancreatic ductal adenocarcinoma (C1). The Supplemental Online Appendix contains the
corresponding pedigree information. The non family controls were denoted with the prefix N
(N1, N2, N3, N4) for controls in the duodenal juice studies. We sampled duodenal juice from the
patients by endoscopy. To enrich for pancreatic factors, we administered intravenous secretin
((Secrelux® Sanofi, Germany. 1 cU/kg, max 70 cU) to the patients and sampled duodenal juice
from 30 to 45 minutes later since it has been shown that there is a peak secretion from pancreas
in this time interval (12; 13). The controls for the secretin stimulated duodenal juice studies were
recruited from volunteers. The single pancreatic juice specimen from a CEL mutation carrier
with PDAC was collected during a classical Whipple procedure. All studies were approved by
the Norwegian regional committee for research ethics and the IRB at the Joslin Diabetes Center
and performed according to the Helsinki Declaration. We obtained written informed consent
from subjects or their parents. Patients were of North European descent and recruited from the
Norwegian MODY Registry.
Protocol for pancreatic imaging. MR imaging was performed on a 1.5 T Siemens Avanto
running Syngo MR B17 (Erlagen, Germany) using a 12 channel body coil. For clinical and
anatomical evaluation of the pancreas a standard abdominal protocol was used: Axial TrueFISP
(true Fast Imaging with Steady state Precision; TR (repetition time) = 2.79 ms, TE (echo time) =
1.17 ms, slice thickness = 4.0 mm, spacing = 0, alfa = 63 degrees, FOV (field of view) = 380 mm
x 380 mm, matrix = 156 x 192, acquisition time = 15 s) and axial fat saturated T2 weighted
4
Diabetes Page 6 of 61
imaging (TR = 1000 ms, TE (echo time) = 90 ms, slice thickness = 5 mm, spacing = 0, alfa = 150 degrees, FOV (field of view) = 350 mm x 350 mm, matrix = 256 x 256, acquisition time = 24 s).
For evaluation of the pancreatic and biliary ducts magnetic resonance cholangiopancreaticography (MRCP) was performed: Coronal T2 weigthed imaging (TR = 2000 ms, TE (echo time) = 691 ms, slice thickness = 1.1 mm, spacing = 0, alfa = 140 degrees, FOV
(field of view) = 280 mm x 280 mm, matrix = 320 x 320, acquisition time = 5.23 min) with maximum intensity projection (MIP) and multiplanar reformatting (MPR).
Cytokine assay. Forty two cytokines were measured in secretin stimulated duodenal juice samples (sample volume of 25 microliter), using the MILLIPLEX MAP Human
Cytokine/Chemokine Premixed 42 Plex kit from Millipore (Billerica, MA). This 42 plex kit was used to measure the concentrations of EGF, Eotaxin, FGF 2, Flt 3 ligand, Fractalkine, G
CSF, GM CSF, GRO, IFN α2, IFN γ, IL 10, IL 12 (p40), IL 12 (p70), IL 13, IL 15, IL 17, IL
1Rα, IL 1α, IL 1β, IL 2, IL 3, IL 4, IL 5, IL 6, IL 7, IL 8, IL 9, IP 10, MCP 1, MCP 3, MDC
(CCL22), MIP 1α, MIP 1β, PDGF AA, PDGF AB/BB, RANTES, TGF α, TNF α, TNF β,
VEGF, sCD40L, sIL 2Rα. All samples were analyzed with a Luminex 200 xMAP system, and
MILLIPLEX Analyst Software (VigeneTech, Carlisle, MA) was used to analyse the results.
Proteolytic degradation assay. A set of 45 synthetic peptide substrates, each at 1 M, were incubated with 10 g pancreatic fluid and reaction buffer containing Tris Cl (25 mM, pH 7.5),
MgCl2 (7.5 mM), EGTA (0.2 mM), β glycerophosphate (7.5 mM), Na3VO4 (0.1 mM) and DTT
(0.1 mM). The reaction was incubated at room temperature for 0, 15, 60 min, after which the reaction was quenched with 100 L of 1% trifluoroacetic acid (TFA). Twenty five pmol of internal standard stable isotope labeled substrate peptides was spiked into the quenched reaction.
The solution was desalted using Sep Pak C18 50 mg cartridges (Waters) and dried using vacuum
5
Page 7 of 61 Diabetes
centrifugation. The desalted sample was then resuspended in 100 L of 5% formic acid of which
2 L was then subjected to analysis by mass spectrometry. Technical duplicates were performed
for each sample at each time point. The peptides from proteolytic degradation assays were
analysed by LC MS on a high resolution Exactive Orbitrap mass spectrometer (Thermo). Briefly,
a 45 minute gradient was used to separate the peptides from 10 37% solvent B (0.125% formic
acid in acentonitrile) at a flow rate of 300 mL/min. LC MS data was collected from 350 1500
m/z and the extracted ion chromatograms of the light and heavy peptides were used to quantitate
the absolute kinase activity and the extent of peptide degradation respectively. Data analysis was
performed using Pinpoint (Thermo).
Quantitative proteomics of pancreatic juice using TMT labeling. Pancreatic juice (100 L)
from 6 subjects was purified using methanol/chloroform precipitation. The purified protein
pellets were each resuspended in 100 L of 50 mM HEPES, 4M Urea, pH 8.5. The protein
concentration was determined using Bradford protein assay (Pierce). The cysteine residues were
reduced and alkylated with DTT (5 mM) and iodoacetamide (15 mM) respectively prior to
digestion with Lys C (Wako, Japan) in a 1:100 LysC:protein ratio. Following digestion
overnight, the digest was acidified with formic acid prior to desalting with C18 solid phase
extraction (Sep Pak, Waters, Milford, MA). The desalted peptides were then resuspended in 100
L of 50 mM HEPES, pH 8.5 and each sample was labeled with one of the 6 plex tandem mass
tag (TMT) reagents (Thermo Scientific, Rockford, IL) for one hour at room temperature. The
TMT labeled peptides were purified, fractionated by strong cation exchange chromatography
(SCX) and 14 fractions were analysed by LC MS/MS on an LTQ Orbitrap Velos mass
spectrometer. An MS3 method developed recently by the lab was used to overcome the
interference problem in acquisition of TMT data as described (14). Further experimental details
for the proteomics and immunoblotting experiments, kinase activity assay (15) and proteolytic
6
Diabetes Page 8 of 61
degardation assays are outlined in the Supplemental Online Appendix. Details for the database searches, filtering and quantification for the proteomics experiments are also outlined in the
Supplemental Online Appendix, but, briefly, two different types of searches (LysC and "no enzyme") were used when MS/MS spectra were searched against the human ipi database (v. 3.6) using the Sequent algorithm (v. 5) (16). The data was filtered to a false discovery rate of less than
1% based on the target decoy database approach (17). Further filtering of the list to include only
'pancreas specific' proteins was based on analyses of nine different mouse tissues (18). To test the hypothesis that secretin stimulated duodenal secretory proteins truly reflected pancreatic disease, an investigator (F.M.) blinded to the sample status, performed unsupervised clustering analysis based on protein abundance (i.e. algoritmic classification procedure to group samples based on protein expression similarities and blinded to a knowledge of sample disease status).
Bioinformatics studies using Biobase. We performed a comprehensive investigation of all proteins in the secretin stimulated duodenal juice (not only pancreatic specific proteins) to avoid excluding proteins that are only expressed in the diseased state. We used the Biobase Explain 3.0 tool (19) to examine whether the proteins that were elevated in the secretin stimulated duodenal juice from CEL mutation carriers (log2 ratio>1 of protein expression) shared common upstream key nodes in pathways in physiological and pathophysiological processes, see http://explain30.biobase international.com.ezp prod1.hul.harvard.edu/cgi bin/biobase/ExPlain_3.0/bin/start.cgi? for details.
Other statistical analysis. For the analyses of cytokine levels we used two tailed Student’s t tests of independent groups assuming unequal variance. We used linear regression to estimate R2 for the correlation of cyst number and the respective cytokines. We chose a significance level of
7
Page 9 of 61 Diabetes
5% and analysed all data using Stata 11.0 (Stata Statistical Software, Stata Corp., College
Station, Texas, USA).
RESULTS
Subjects with CEL-MODY Develop Multiple Pancreatic Cysts and diabetes in their forties.
We performed pancreatic imaging studies in a CEL MODY family (Figure 1) and identified
multiple unilocular pancreatic cysts (Figure 2) in all CEL mutation carriers (n=8) who had also
developed diabetes (Table 1). None of the non diabetic CEL mutation carriers (n=4) or the
healthy controls (n=6) had multiple pancreatic cysts, but two of these subjects (one non diabetic
CEL mutation carrier and a healthy control) had a solitary cyst. Moreover, the number of cysts
correlated positively with age in the CEL mutation carriers (R2 = 0.46, P = 0.01).
Cytokine analysis of the secretin-stimulated duodenal juice identifies MAPK-driven
cytokines in CEL mutation carriers positively correlated with disease progression. Since
cytokine levels in secretin stimulated duodenal juice may reflect pancreatic disease (20), we first
assessed a panel of 42 cytokines in the secretin stimulated duodenal fluid samples of several
family members and controls (Suppl. Table 1). The MAPK driven CXCR2 receptor targeting
cytokine growth related oncogene (GRO) was significantly increased in the carriers with a
tendency towards higher levels in subjects who had accumulated more clinical manifestations
(Table 1, and Fig. 3a; P<0.01). We also observed that GRO correlated significantly with the
number of cysts in the subjects (Fig. 3b; R2=0.56, P=0.005). Interestingly, the related MAPK
driven CXCR1 and 2 targeting cytokine interleukin 8 (IL8), while not showing significantly
increased levels in CEL mutation carriers (Fig. 3c; P=0.15), revealed a significant correlation
with the number of cysts (Fig. 3d; R2=0.72, P<0.001; Suppl. Table 1). Hence, secretin stimulated
8
Diabetes Page 10 of 61
duodenal fluid of CEL mutation carriers contains cytokines whose increasing levels may potentially reflect increasing severity of a progressive pancreatic disease.
Proteomics-based identification of a distinct protein signature in secretin-stimulated duodenal juice of CEL mutation carriers. Since some protein markers (cytokines) displayed increasing levels with the progressive accumulation of clinical manifestations, we asked whether diabetic CEL mutation carriers displayed a unique proteomic signature in secretin stimulated duodenal juice. Anticipating some degree of protein degradation in duodenal juice we first assessed the extent of protein degradation by exploring a multiplexed mass spectrometry based degradation assay (Fig. 4a) and differences in silver staining for equal quantities of protein loaded onto gels (Fig. 4b) and found indeed an extensive degradation of secretin stimulated duodenal proteins. Subsequently, we applied a multiplexed quantitative proteomics approach with isobaric labeling (TMT, tandem mass tags) to a training set of secretin stimulated duodenal juice from three CEL mutation carriers and three healthy controls. Consistent with the previously identified protein degradation we found large differences in protein identifications using standard
LysC compared to ‘no enzyme’ specific searches of proteomics data (Fig 4c). Hence, to identify both the degraded and non degraded proteins to account for all the secreted proteins in the original sample, we further pursued no enzyme specified database searching, and we identified
757 proteins (1% false discovery rate) in the secretin stimulated duodenal juice (Suppl. Table 2) despite the extensive protein degradation. Further filtering of the list to include only proteins abundantly expressed in mice pancreata (18) resulted in the identification of 115 proteins
(excerpt in Table 2). Intriguingly, an unsupervised clustering analysis clearly separated the patients and the controls into two separate groups (Fig 5a), supporting the existence of a unique proteomic signature (Table 2 and Suppl. Table 2) in diabetic CEL mutation carriers. We identified mostly digestive enzymes (Table 2) among the duodenal proteins showing a relatively
9
Page 11 of 61 Diabetes
higher abundance in control samples (Fig. 5b), including carboxyl ester lipase (CEL) and
elastase (CELA2A, CELA3B), consistent with our anticipation of a greater abundance of
pancreatic proteases in healthy subjects. Proteins more abundant in CEL mutation carriers (Table
2) included proteins associated with diabetes (TSPAN8; (21)) and insulin secretion
(GLUD1;(22)). Together these observations provide confidence that secretin stimulated duodenal
proteins actually reflect pancreatic output and could serve as potential indicators of pancreatic
pathology.
Validation of the training set proteins by immunoblotting and their potential role as
mitogen-activated protein kinase (MAPK) targets. Next, to validate the proteome signature in
CEL mutation carriers, we applied immunoblotting of selected signature proteins in secretin
stimulated duodenal juice from the training set of three CEL mutation carriers and three controls.
Indeed, band intensities and mass spectrometry abundances correlated well for the proteins
(compare Fig. 5c and Suppl. Table 2), confirming the validity of the mass spectrometry findings.
Furthermore, band intensities were also clearly different between controls and two additional pre
diabetic CEL mutation carriers (Fig. 5c). Notably, the protein markers LMNA, SKP1 and KRT18
clearly separated CEL mutation carriers from controls, whereas the protein markers PKM2 and
GSN more partially separated these groups. In order to assess whether the proteome signature
proteins in CEL mutation carriers were parts of an activated pathway, we explored the potential
connectivity of the top 30 most abundant proteins in the training set using a curated database
search (Biobase Explain) of reported protein interactions (19). Intriguingly, we found that several
of these validated markers were downstream targets of MAPK (Fig. 5d and Suppl. Table 3),
including several of the proteins also validated by immunoblotting (compare Fig. 5d with Fig.
5c).
10
Diabetes Page 12 of 61
To further assess the strength of connectivity between the most abundant proteins, we used Gene
Network Enrichment Analysis (GNEA; (23; 24)) to test for significant protein interactions based on yeast two hybrid systems data. We identified the 14 3 3 protein zeta, encoded by YWHAZ, interacting with Keratin 18 (KRT18) and the subnetwork of interacting proteins around YWHAZ to be cumulatively significant (Fisher’s p = 0.00028), supporting interactions between 14 3 3 protein zeta and keratin 18 in disease pathogenesis (Suppl. Fig. 1). Both these proteins were also
MAPK targets as defined by the Biobase Explain findings.
Multiplexed kinase studies provide further evidence of altered kinase activity. Since the above data suggested the involvement of mitogen activated protein kinase signaling we profiled multiple kinase activities in both duodenal samples using mass spectrometry ((15); Fig. 5e).
Upon examining a different aliquot of the same secretin stimulated duodenal juice samples as that used in the immunoblotting studies, and adopting unsupervised clustering analysis we observed that the three controls and the pre diabetic CEL mutation carrier clustered in one group clearly different from a group with the four diabetic CEL mutation carriers (including a subject with pancreatic ductal adenocarcinoma). Interestingly, the cell cycle related kinases, CHK1 and
CHK2, and PKC, and to a lesser extent also MAPK, displayed elevated kinase activity in the diabetic CEL mutation carriers (Fig. 5f).
DISCUSSION
Our data further support that CEL MODY is a progressive disease. We have previously reported the occurence of pancreatic exocrine dysfunction from infancy (3), pancreatic lipomatosis form early childhood (4) and diabetes developing in the forties (3). Here, we show that CEL mutation carriers also develop multiple pancreatic cysts as they develop diabetes. Thus, there seems to be a
11
Page 13 of 61 Diabetes
sequence of clinical characteristics over time that may reflect a pathogenic process taking place
in the pancreas and ultimately leading to diabetes. Our data support that this pathogenic process
is associated with upregulated MAPK signaling for the following reasons: First, cytokine
analyses in the pancreatic secretome demonstrated increasing levels of the MAPK driven (25)
cytokine GRO over the course of the disease. Second, using a sensitive and accurate quantitative
proteomics approach, we identified elevated levels of downstream target proteins of
MAPK/ERK2 signaling in diabetic CEL mutation carriers. Third, the mass spectrometry data
were confirmed by immunoblotting for proteins in the duodenal juice, and immunoblotting also
confirmed our observations in several other CEL mutation carrying subjects. Finally, we
identified elevated kinase activity including that of MAPK in CEL mutation carriers.
Our data also support our hypothesis that secretin stimulated duodenal secretory proteins truly
reflect pancreatic disease. First, unsupervised clustering analysis of proteomics data correctly
identified the diseased subjects. Second, we also detected increased levels of digestive enzymes
in healthy subjects consistent with our anticipation of a greater abundance of pancreatic proteases
in healthy subjects and consistent with proteomics findings in normal pancreatic juice by ERCP
(26), by enzymatic findings (of elastase) in feces (3) and by enzymatic findings (of amylase,
lipase and chymoptrypsin) in the secretin stimulated duodenal juice of CEL mutation carriers
(27). Third, the increased levels of digestive enzymes were confirmed by immunoblotting (for
amylase).
We have described a new feature of the CEL MODY phenotype by demonstrating the presence
of pancreatic cysts in mutation carriers. Such cysts are an established risk factor for pancreatic
cancer (28). Notably, one of the oldest affected members of the family was diagnosed with
pancreatic ductal adenocarcinoma at the age of 78. We therefore conclude that CEL MODY
12
Diabetes Page 14 of 61
patients could be at increased risk for developing pancreatic cancer. The cytokine analyses revealed a linear relationship between the number of cysts and the CXCR1 and CXCR2 targeting
MAPK driven (25) cytokines (GRO, IL8); a finding consistent with a previous report of elevated
IL8 levels in sera of patients with pancreatic cancer (29). We believe that the low levels of GRO and IL8 despite multiple cysts in a diabetic CEL mutation carrier (Subject D1) fits with the long duration of his diabetes and the development of end stage pancreatic disease with loss of inflamatory cells as observed in end stage pancreatitis (30), although there also the possibility of phenotypic variance within the family. The finding of MAPK related proteins and cytokines in duodenal juice of subjects with progressive pancreatic disease is particularly interesting due to the role of MAPK both in diabetes and cancer (31). In the subject with cancer, the tumor had a somatic KRAS mutation (G12V, not shown) commonly observed in pancreatic adenocarcinomas
(32). Cancer associated KRAS mutations generally lead to overactive proteins that stimulate oncogenic signaling through the MAPK pathway (33).
In conclusion, subjects with CEL MODY develop multiple pancreatic cysts and diabetes in their forties. Increased levels of MAPK target proteins may reflect the pathophysiological development of pancreatic cysts and diabetes in CEL MODY. These data suggest that the MAPK pathway should be further explored in subjects with CEL MODY in order to find drug targets for the possible prevention of disease development.
ACKNOWLEDGEMENTS
This work was supported by NIH grant RO1 DK 67536 and the Graetz Bridge Funds (to R.N.
Kulkarni), and the Joslin DRC Specialized Assay and Advanced Microscopy Cores (NIH P30
13
Page 15 of 61 Diabetes
DK36836) and NIH grant HG3456 (to S.P.G.) and the Norwegian Research Council (to H.R. and
P.R.N.) and by grants from the Western Norway Regional Health Authority (to H.R., H.I., and
P.R.N.) and the Norwegian Endocrine Society (to H.R.) and the Norwegian Cancer Society (to
A.M.) and the Fulbright Foundation (to H.R.), and the University of Bergen (to P.R.N.), and
Innovest (to P.R.N.).
No potential conflicts of interest relevant to this article were reported.
HR SPG RNK designed the study; HR FEMA ET SB IH JH SMW MV AEO DH GD collected
the data; HR FEMA ET SB IH ML MBR HI PRN AM SPG RNK contributed to data analysis
and interpretation. The manuscript was drafted by HR and RNK with input from all authors. All
authors contributed to the revision and approved the final version of the manuscript.
HR and RNK are the guarantors of this work and, as such, had full access to all data in the study
and take responsibility for the integrity of the data and accuracy of the data analysis.
The authors thank C. Ronald Kahn MD and Chong Wee Liew PhD of Joslin Diabetes Center for
discussions; G. Sankaranarayanan of Joslin Diabetes Center for assistance with cytokine assays;
C. Cahill of Joslin Diabetes Center for assistance with electron microscopy; E. Huttlin of
Harvard Medical School for assistance with data analysis of pancreatic specific proteins.
REFERENCES
1. Pearson ER, Starkey BJ, Powell RJ, Gribble FM, Clark PM, Hattersley AT: Genetic cause of hyperglycaemia and response to treatment in diabetes. Lancet 2003;362:1275 1281 2. Ashcroft FM, Rorsman P: Diabetes mellitus and the beta cell: the last ten years. Cell 2012;148:1160 1171 3. Ræder H, Johansson S, Holm PI, Haldorsen IS, Mas E, Sbarra V, Nermoen I, Eide SA, Grevle L, Bjørkhaug L, Sagen JV, Aksnes L, Søvik O, Lombardo D, Molven A, Njølstad PR: Mutations in the CEL VNTR cause a syndrome of diabetes and pancreatic exocrine dysfunction. Nat Genet 2006;38:54 62 4. Ræder H, Haldorsen IS, Ersland L, Gruner R, Taxt T, Søvik O, Molven A, Njølstad PR: Pancreatic lipomatosis is a structural marker in nondiabetic children with mutations in carboxyl ester lipase. Diabetes 2007;56:444 449
14
Diabetes Page 16 of 61
5. Lombardo D: Bile salt dependent lipase: its pathophysiological implications. Biochim Biophys Acta 2001;1533:1 28 6. Vesterhus M, Ræder H, Kurpad AJ, Kawamori D, Molven A, Kulkarni RN, Kahn CR, Njølstad PR: Pancreatic Function in Carboxyl Ester Lipase Knockout Mice. Pancreatology 2010;10:467 476 7. Ræder H, Vesterhus M, El Ouaamari A, Paulo JA, McAllister FE, Liew CW, Hu J, Kawamori D, Molven A, Gygi SP, Njølstad PR, Kahn CR, Kulkarni RN: Absence of Diabetes and Pancreatic Exocrine Dysfunction in a Transgenic Model of Carboxyl Ester Lipase MODY (Maturity Onset Diabetes of the Young). PLoS One 2013;8:e60229 8. Johansson BB, Torsvik J, Bjørkhaug L, Vesterhus M, Ragvin A, Tjora E, Fjeld K, Hoem D, Johansson S, Ræder H, Lindquist S, Hernell O, Cnop M, Saraste J, Flatmark T, Molven A, Njølstad PR: Diabetes and pancreatic exocrine dysfunction due to mutations in the carboxyl ester lipase gene maturity onset diabetes of the young (CEL MODY): a protein misfolding disease. J Biol Chem 2011;286:34593 34605 9. Paulo JA, Lee LS, Wu B, Banks PA, Steen H, Conwell DL: Mass spectrometry based proteomics of endoscopically collected pancreatic fluid in chronic pancreatitis research. Proteomics. Clinical applications 2011;5:109 120 10. Ting L, Rad R, Gygi SP, Haas W: MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics. Nature methods 2011; 11. Kunz RC, McAllister FE, Rush J, Gygi SP: A high throughput, multiplexed kinase assay using a benchtop orbitrap mass spectrometer to investigate the effect of kinase inhibitors on kinase signaling pathways. Anal Chem 2012;84:6233 6239 12. Stevens T, Conwell DL, Zuccaro G, Jr., Lewis SA, Love TE: The efficiency of endoscopic pancreatic function testing is optimized using duodenal aspirates at 30 and 45 minutes after intravenous secretin. The American journal of gastroenterology 2007;102:297 301 13. Stevens T, Conwell DL, Zuccaro G, Jr., Vargo JJ, Dumot JA, Lopez R: Comparison of endoscopic ultrasound and endoscopic retrograde pancreatography for the prediction of pancreatic exocrine insufficiency. Digestive diseases and sciences 2008;53:1146 1151 14. Ting L, Rad R, Gygi SP, Haas W: MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics. Nat Methods 2011;8:937 940 15. Kubota K, Anjum R, Yu Y, Kunz RC, Andersen JN, Kraus M, Keilhack H, Nagashima K, Krauss S, Paweletz C, Hendrickson RC, Feldman AS, Wu CL, Rush J, Villen J, Gygi SP: Sensitive multiplexed analysis of kinase activities and activity based kinase identification. Nat Biotechnol 2009;27:933 940 16. Eng JK, McCormack AL, Yates JR, 3rd: An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom. 1994;5:976 989 17. Elias JE, Gygi SP: Target decoy search strategy for increased confidence in large scale protein identifications by mass spectrometry. Nature methods 2007;4:207 214 18. Huttlin EL, Jedrychowski MP, Elias JE, Goswami T, Rad R, Beausoleil SA, Villen J, Haas W, Sowa ME, Gygi SP: A tissue specific atlas of mouse protein phosphorylation and expression. Cell 2010;143:1174 1189 19. Kel A, Voss N, Valeev T, Stegmaier P, Kel Margoulis O, Wingender E: ExPlain: finding upstream drug targets in disease gene regulatory networks. SAR and QSAR in environmental research 2008;19:481 494 20. Paulo JA, Lee LS, Wu B, Banks PA, Steen H, Conwell DL: Cytokine profiling of pancreatic fluid using the ePFT collection method in tandem with a multiplexed microarray assay. J Immunol Methods 2011;369:98 107 21. Craddock N, Hurles ME, Cardin N, Pearson RD, Plagnol V, Robson S, Vukcevic D, Barnes C, Conrad DF, Giannoulatou E, Holmes C, Marchini JL, Stirrups K, Tobin MD, Wain LV, Yau
15
Page 17 of 61 Diabetes
C, Aerts J, Ahmad T, Andrews TD, Arbury H, Attwood A, Auton A, Ball SG, Balmforth AJ, Barrett JC, Barroso I, Barton A, Bennett AJ, Bhaskar S, Blaszczyk K, Bowes J, Brand OJ, Braund PS, Bredin F, Breen G, Brown MJ, Bruce IN, Bull J, Burren OS, Burton J, Byrnes J, Caesar S, Clee CM, Coffey AJ, Connell JM, Cooper JD, Dominiczak AF, Downes K, Drummond HE, Dudakia D, Dunham A, Ebbs B, Eccles D, Edkins S, Edwards C, Elliot A, Emery P, Evans DM, Evans G, Eyre S, Farmer A, Ferrier IN, Feuk L, Fitzgerald T, Flynn E, Forbes A, Forty L, Franklyn JA, Freathy RM, Gibbs P, Gilbert P, Gokumen O, Gordon Smith K, Gray E, Green E, Groves CJ, Grozeva D, Gwilliam R, Hall A, Hammond N, Hardy M, Harrison P, Hassanali N, Hebaishi H, Hines S, Hinks A, Hitman GA, Hocking L, Howard E, Howard P, Howson JM, Hughes D, Hunt S, Isaacs JD, Jain M, Jewell DP, Johnson T, Jolley JD, Jones IR, Jones LA, Kirov G, Langford CF, Lango Allen H, Lathrop GM, Lee J, Lee KL, Lees C, Lewis K, Lindgren CM, Maisuria Armer M, Maller J, Mansfield J, Martin P, Massey DC, McArdle WL, McGuffin P, McLay KE, Mentzer A, Mimmack ML, Morgan AE, Morris AP, Mowat C, Myers S, Newman W, Nimmo ER, O'Donovan MC, Onipinla A, Onyiah I, Ovington NR, Owen MJ, Palin K, Parnell K, Pernet D, Perry JR, Phillips A, Pinto D, Prescott NJ, Prokopenko I, Quail MA, Rafelt S, Rayner NW, Redon R, Reid DM, Renwick, Ring SM, Robertson N, Russell E, St Clair D, Sambrook JG, Sanderson JD, Schuilenburg H, Scott CE, Scott R, Seal S, Shaw Hawkins S, Shields BM, Simmonds MJ, Smyth DJ, Somaskantharajah E, Spanova K, Steer S, Stephens J, Stevens HE, Stone MA, Su Z, Symmons DP, Thompson JR, Thomson W, Travers ME, Turnbull C, Valsesia A, Walker M, Walker NM, Wallace C, Warren Perry M, Watkins NA, Webster J, Weedon MN, Wilson AG, Woodburn M, Wordsworth BP, Young AH, Zeggini E, Carter NP, Frayling TM, Lee C, McVean G, Munroe PB, Palotie A, Sawcer SJ, Scherer SW, Strachan DP, Tyler Smith C, Brown MA, Burton PR, Caulfield MJ, Compston A, Farrall M, Gough SC, Hall AS, Hattersley AT, Hill AV, Mathew CG, Pembrey M, Satsangi J, Stratton MR, Worthington J, Deloukas P, Duncanson A, Kwiatkowski DP, McCarthy MI, Ouwehand W, Parkes M, Rahman N, Todd JA, Samani NJ, Donnelly P: Genome wide association study of CNVs in 16,000 cases of eight common diseases and 3,000 shared controls. Nature 2010;464:713 720 22. Stanley CA, Lieu YK, Hsu BY, Burlina AB, Greenberg CR, Hopwood NJ, Perlman K, Rich BH, Zammarchi E, Poncz M: Hyperinsulinism and hyperammonemia in infants with regulatory mutations of the glutamate dehydrogenase gene. N Engl J Med 1998;338:1352 1357 23. Strnad P, Tao GZ, Zhou Q, Harada M, Toivola DM, Brunt EM, Omary MB: Keratin mutation predisposes to mouse liver fibrosis and unmasks differential effects of the carbon tetrachloride and thioacetamide models. Gastroenterology 2008;134:1169 1179 24. Tao GZ, Li DH, Zhou Q, Toivola DM, Strnad P, Sandesara N, Cheung RC, Hong A, Omary MB: Monitoring of epithelial cell caspase activation via detection of durable keratin fragment formation. J Pathol 2008;215:164 174 25. Ancrile BB, O'Hayer KM, Counter CM: Oncogenic ras induced expression of cytokines: a new target of anti cancer therapeutics. Molecular interventions 2008;8:22 27 26. Zhou L, Lu Z, Yang A, Deng R, Mai C, Sang X, Faber KN, Lu X: Comparative proteomic analysis of human pancreatic juice: methodological study. Proteomics 2007;7:1345 1355 27. Tjora E, Wathle G, Engjom T, Erchinger F, Molven A, Aksnes L, Haldorsen IS, Dimcevski G, Njolstad PR, Raeder H: Severe Pancreatic Dysfunction But Compensated Nutritional Status in Monogenic Pancreatic Disease Caused by Carboxyl Ester Lipase Mutations. Pancreas 2013; 28. Tanaka S, Nakao M, Ioka T, Takakura R, Takano Y, Tsukuma H, Uehara H, Suzuki R, Fukuda J: Slight dilatation of the main pancreatic duct and presence of pancreatic cysts as predictive signs of pancreatic cancer: a prospective study. Radiology 2010;254:965 972 29. Wigmore SJ, Fearon KC, Sangster K, Maingay JP, Garden OJ, Ross JA: Cytokine regulation of constitutive production of interleukin 8 and 6 by human pancreatic cancer cell lines and serum cytokine concentrations in patients with pancreatic cancer. Int J Oncol 2002;21:881 886
16
Diabetes Page 18 of 61
30. Braganza JM, Lee SH, McCloy RF, McMahon MJ: Chronic pancreatitis. Lancet 2011;377:1184 1197 31. Lawrence MC, Jivan A, Shao C, Duan L, Goad D, Zaganjor E, Osborne J, McGlynn K, Stippec S, Earnest S, Chen W, Cobb MH: The roles of MAPKs in disease. Cell Res 2008;18:436 442 32. Immervoll H, Hoem D, Kugarajh K, Steine SJ, Molven A: Molecular analysis of the EGFR RAS RAF pathway in pancreatic ductal adenocarcinomas: lack of mutations in the BRAF and EGFR genes. Virchows Arch 2006;448:788 796 33. Collisson EA, Trejo CL, Silva JM, Gu S, Korkola JE, Heiser LM, Charles RP, Rabinovich BA, Hann B, Dankort D, Spellman PT, Phillips WA, Gray JW, McMahon M: A central role for RAF >MEK >ERK signaling in the genesis of pancreatic ductal adenocarcinoma. Cancer Discov 2012;2:685 693
17
Page 19 of 61 Diabetes
TABLES
Largest Fecal Diabetes Age Number cyst Pancreatic GRO IL8 Status Sex elastase Diabetes? duration (years) of cysts diameter duct>2.5 (pg/mL) (pg/mL) (ug/g) (years) (mm) mm? Control F 32 426 no 0 0 0 no 2.5 0.3 Control (N2) F 38 561 no 0 0 0 no 7.3 0.0 Control M 40 584 no 0 0 0 no 5.9 1.5 Control F 50 723 no 0 0 0 no 0.0 0.2 Control F 53 486 no 0 1 2 no 2.5 0.1 Control F 64 525 no 0 0 0 no 9.7 2.1 Control (N1) M 63 363 no 0 NA NA NA 4.3 2.6 Control (N3) M 25 299 no 0 NA NA NA 11.3 4.4 CEL carrier M 9 448 no 0 0 0 no 2.5 2.0 CEL carrier M 14 50 no 0 0 0 no 11.1 4.5 CEL carrier F 17 71 no 0 1 6 no 5.1 5.3 CEL carrier M 37 10 no 0 0 0 (P2) no NA NA CEL carrier M 40 12 no 0 0 0 (P1) no 7.3 7.7 CEL carrier F 43 0 yes 13.1 12 7 (D2) no 116.7 21.1 CEL carrier F 45 51 yes 6.2 9 4 no 45.1 16.8 CEL carrier M 56 0 yes 11.8 6 4 no 97.3 4.3 CEL carrier M 57 0 yes 17.5 4 11 yes 15.2 3.1 CEL carrier F 57 5 yes 19.1 60 11 (D4) no 109.7 373.6 CEL carrier M 65 0 yes 44.7 20 13 (D1) no 2.5 1.2 CEL carrier M 69 3 yes 13.6 30 9 (D3) no 88.8 4.4 CEL carrier 60 13 F 78 0 yes 37.5 (C1) no 161.0 167.7
Table 1: Clinical characteristics of CEL mutation carriers and controls. NA, not available.
The alphanumeric codes i Status are explained in Methods.
18
Diabetes Page 20 of 61
Gene ID Log 2 ratio Log 2 total Gene ID Log 2 ratio Log 2 total 1 CLEC3B 16.0 23.2 87 MUC1 2.4 20.2 2 KRT18 15.2 22.4 88 CUZD1 2.5 25.8 3 NUCB2 12.5 19.7 89 CLPS 2.6 22.5 4 DCI 11.5 19.6 90 PNLIPRP2 2.7 27.3 5 MYL1 11.5 18.7 91 KLK1 2.7 24.1 6 RPLP2 11.1 23.2 92 RNASE1 3.5 25.7 7 SKP1 10.8 18.1 93 CPA2 3.6 27.7 8 RPLP0 10.7 21.7 94 S100A7 3.9 22.4 9 RPLP1 9.7 21.4 95 CEL 3.9 28.5 10 SFN 9.5 19.1 96 CTRB1 3.9 27.8 11 EPB41L3 9.4 22.1 97 CTRL 3.9 23.9 12 CHGA 8.9 16.1 98 PRSS1 4.0 28.1 13 PKM2 7.5 22.3 99 CPA1 4.1 28.9 14 IDH1 7.1 22.5 100 CELA3B 4.3 25.6 15 MYH14 6.2 22.3 101 PNLIPRP1 4.3 26.3 16 PURA 6.2 20.7 102 IPI00219910 4.6 23.5 17 LMNA 5.5 21.3 103 PNLIP 4.8 30.0 18 KRT8 5.1 24.9 104 PRSS2 4.8 26.6 19 EEF1A1 5.1 21.0 105 CELA3A 4.9 27.8 20 GLUD1 4.9 22.5 106 CTRC 5.0 26.8 21 PCDH24 4.7 21.8 107 GP2 5.2 25.8 22 PPA1 4.1 22.1 108 CPB1 5.3 27.9 23 NPC2 4.0 20.2 109 AMY1C 5.3 26.8 24 ALDOB 3.8 25.5 110 AMY2B 5.4 29.6 25 TSPAN8 3.7 20.2 111 SERPINI2 6.7 22.8 26 CLIC1 3.7 23.0 112 PLA2G1B 6.9 25.8 27 PGD 3.7 19.9 113 CD302 8.2 16.7 28 CST3 3.3 23.5 114 HPN 8.4 18.4 29 GSN 3.3 24.9 115 CTRB2 11.3 18.5
Table 2: The most extreme differentially expressed proteins in secretin-stimulated duodenal juice. Log2 ratio, Log2 of the ratio of mass spectrometry abundance for the sum of the three healthy controls divided by the sum of the three diabetic mutation carriers. Log 2 total, Log 2 of the total mass spectrometry abundance for a protein as the sum of the abundances of three healthy controls and the three diabetic mutation carriers.
19
Page 21 of 61 Diabetes
FIGURE LEGENDS
Fig. 1: Overview of the multimodal systems biology approach.
A systems biology approach using secretin stimulated duodenal juice from subjects in a CEL
MODY family to discover early markers in pancreatic disease development by proteomics
methods.
Fig. 2: The Magnetic Resonance Imaging of Pancreatic cysts in CEL mutation carriers.
MRCP (a c) and axial fat saturated T2 weighted imaging at the level of the pancreatic body (d f)
in two CEL mutation carriers (a, d and b, e) and a normal control (c, f). Both mutation carriers
had multiple cystic lesions within the pancreatic body and tail (white thin arrows; a b, d e) as
well as in the pancreatic head (arrowheads; a b). The pancreatic ducts (filled black arrows) and
common bile ducts (filled white arrows) have normal caliper (a b, d e) similar to that in the
control (c, f). GB, gallbladder. The alphanumeric codes refer to the subject designation explained
in Research Design and Methods.
Fig. 3: The identification of a patient-specific cytokine signature in pancreatic juice and a
mass spectrometry degradation assay.
Cytokine levels for GRO compared to mutation status (a) and correlated with cyst number (b).
Cytokine levels for IL 8 compared to mutation status (c) and correlated with cyst number (d).
Further, IL 1b was increased in the carriers (P=0.005) and also significantly correlated with the
number of cysts (R2=0.70, P<0.001). None of the remaining cytokines correlated significantly
20
Diabetes Page 22 of 61
with the number of cysts although significantly elevated in the carriers (FGF 2 (P=0.04), Flt3 ligand (P=0.03), GM CSF (P=0.01), IL 1ra (P=0.02), IL 12 (P=0.01), MCP 1(P=0.04), s CD
40L (P=0.03); Suppl. Table 1).
Fig. 4: Reduced substrate degradation in CEL-MODY patients.
Greater substrate degradation was observed for control patients compared to CEL MODY patients. A set of 45 substrate peptides are incubated with pancreatic juice for 45 minutes and the amount of degradation is measured by comparison of the substrate with internal standard heavy peptides at 0, 15, 60 minute time points (a). The differences in protein identifications using silver staining of secretin stimulated duodenal juice for equal quantities of protein loaded onto gels (b).
Consistent with this reduced substrate degradation in the secretin stimulated duodenal juice of
CEL MODY patients we also identified differences in protein identifications using LysC and
‘no enzyme’ specific searches of proteomics data (c). Using ‘no enzyme’ specific searches we identified 757 proteins in the secretin stimulated duodenal juice.
Fig. 5: The identification of a patient-specific protein signature in pancreatic juice
Protein quantitation was performed using isobaric labeling with tandem mass tags (TMT) and
LC MS/MS analysis on an LTQ Orbitrap Velos mass spectrometer. An unsupervised clustering analysis identified clearly different expression patterns in CEL mutation carriers compared to controls with analyses performed by an investigator (FM) blinded to the samples (a). Log2 plot of secretin stimulated duodenal proteins protein abundances and ratios of levels in the three CEL mutation carriers compared to the three controls (b). Log2 ratio, Log2 of the ratio of mass spectrometry abundance for the sum of the three controls divided by the sum of the three diabetic mutation carriers. Log 2 total, Log 2 of the total mass spectrometry abundance for a protein as the sum of the abundances of three controls and the three diabetic mutation carriers. Note the
21
Page 23 of 61 Diabetes
lack of skewed distribution of pancreatic proteins in the log2 plot indicating that a majority of the
proteins had been detected despite degradation. Using ‘no enzyme’ specific searches we
identified 757 proteins in the secretin stimulated duodenal juice, of which 115 proteins were
pancreatic specific based on (18). Immunoblot validation examples of proteins identified with
stipled lines indicate gel crops (c). In addition to the three diabetic CEL mutation carriers (D1,
D2, D3) and three non family controls (N1,N2,N3) we also added secretin stimulated duodenal
juice samples from two pre diabetic CEL mutation carriers (subjects P1 and P2) and from the
CEL mutation carrier with the KRAS mutated pancreatic ductal adenocarcinoma (Subject C1).
Visualization of the MAPK targeted proteins identified by Biobase, ExPlain 3.0 tool, and
alphanumeric codes refering to pubmed IDs in Suppl. Table 3 (d). Heatmap demonstrating
differential clustering of kinase activities in the secretin stimulated duodenal juice and pancreatic
tissue of CEL mutation carriers and controls (e). Vertical bar graphs showing significant
differences in kinase activities for PKC, CH1 and CH2 in secretin stimulated duodenal juice and
for PKC in pancreatic tissue. The multiplexed kinase activity assay allows the absolute
quantitation of kinase activities by measuring the amount of phosphorylation of 60 peptide
substrates with known motifs for many kinases. (f) Vertical bar graphs showing differences in
kinase activities for PKC, CH1, CH2 and MAPK in secretin stimulated duodenal juice. PDAC,
pancreatic ductal adenocarcinoma.
22
Diabetes Page 24 of 61
42x9mm (300 x 300 DPI)
Page 25 of 61 Diabetes
110x60mm (300 x 300 DPI)
Diabetes Page 26 of 61
146x116mm (300 x 300 DPI)
Page 27 of 61 Diabetes
152x141mm (300 x 300 DPI)
Diabetes Page 28 of 61
Fig.5 246x235mm (300 x 300 DPI)
Page 29 of 61 Diabetes
Supplemental Online Appendix
Raeder et al; Carboxyl ester lipase Maturity Onset Diabetes of the Young is Associated with Development of Pancreatic Cysts and Upregulated MAPK Signaling in Secretin stimulated duodenal fluid
Supplementary Material
Patients
The subject designation corresponds to the following pedigree status in a published
pedigree (1): D1 = III 9, D2 = IV 11, D3 = III 5, P1 = IV 8, P2 = IV 12.
Sample preparation for tissue samples for kinase activity and proteolytic
degradation assays
Pancreatic tissue was homogenized using a Dounce homogenizer and ice cold lysis
buffer: 10 mM K2HPO4 pH 7.5, 1 mM EDTA, 10 mM MgCl2, 50 mM β
glycerophosphate, 5 mM EGTA, 0.5% Nonidet P 40, 0.1% Brij 35, 0.1% deoxycholic
acid, 1mM sodium orthovanadate, 1mM phenylmethyl sulfonyl fluoride and protease
inhibitors (Complete protease inhibitor tablet, Roche). Following homogenization, the
lysate was centrifuged at 10,000 rpm for 10 min at 4 °C and the supernatant was snap
frozen with liquid nitrogen and stored at 80 °C. Kinase activities were determined on
lysate that had only been frozen once following harvesting since freeze thaw cycles
decrease kinase activities. Protein concentration was determined by the modified
Bradford assay (Pierce).
Proteomic analysis of pancreatic fluid
The purified peptides following digestion were labeled with TMT. Each tube of 0.8
mg for each TMT label was dissolved in 40 L of anhydrous acetonitrile, of which 5
1 Diabetes Page 30 of 61
L of each was used to label peptides from 40 g of pancreatic fluid protein. The labeling reaction was quenched by the addition of 10 l of 5% hydroxylamine and the individual samples combined and desalted with C18 solid phase extraction (Sep Pak,
Waters, Milford, MA).
The combined sample was fractionated by strong cation exchange chromatography
(SCX), similar to that described previously (2). Briefly, a 2.1 x 200 mm polySULFOETHYL A HPLC column (particle size, 5 m; pore size, 200 Å; PolyLC,
Columbia, MD) was used to separate the sample that was resuspended in 300 L SCX buffer A (7 mM KH2PO4, 30% ACN, pH 2.6). A two buffer (SCX A and B) gradient from 0 to 50% SCX buffer B (7 mM KH2PO4, 350 mM KCl, pH 2.6, 30% ACN) in
47 min was used at a flow rate of 0.2 ml/min, followed by 50 to 100% SCX buffer A to buffer B in 4.5 min using an Agilent 1100 quaternary pump with a degasser and a photodiode array detector (PDA) (Thermo Scientific, San Jose, CA). Fractions were collected every 90 s into a 96 well plate, and dried with a speed vac. The dried peptide fractions were then resuspended with 1% FA, and combined into 14 fractions based on the intensity from the SCX chromatographic UV trace and then desalted by
C18 SPE, and dried using a speed vac.
Dried peptides were resuspended in 15 L 4% formic acid, 5% ACN, and 2 L was loaded onto a microcapillary column (100 m I.D, 20 cm, packed with Magic C18AQ resin: 5 m, 100 Å, Michrom Bioresources, Auburn, CA) with Famos autosampler
(LC Packings, Sunnyvale, CA) and an Agilent 1100 binary HPLC pump (Agilent
Technologies, Santa Clara, CA). Reverse phase chromatography was used for peptide separation with a 150 minute gradient of 9 to 32% ACN in 0.125% FA at a flow rate of around 300 nL/min. An LTQ Orbitrap Velos (Thermo Fischer Scientific) was
2 Page 31 of 61 Diabetes
used, with XCalibur 2.0.7 acquisition software. Data acquisition was performed in
data dependent mode with a survey scan in the range of 300 1500 m/z with a 30,000
resolution. The top 10 most intense ions were selected for fragmentation in the LTQ
with a precursor isolation width window of 2 m/z. The AGC target was set at 3x106
for the survey scan and ions were selected for MS2 and their intensity was greater
than 500 accounts. Ions with unassigned or 1+ charge states were excluded. Selected
ions were excluded from further analysis for 30 s. Maximum ion accumulation times
were 1000 ms and 250 ms for survey and MS/MS scans respectively. An MS3 method
developed by the lab was used to overcome the problem of interfering peaks in the
analysis of TMT data. The instrument parameters used were the same as those
described (3). Briefly, after each MS2 analysis, the most intense fragment ion in an
m/z range between 110 160% of the precursor m/z was selected for HCD MS3. The
fragment ion isolation width was set to 4 m/z, the MS3 AGC was 20 x 104 and the
maximum ion time set to 250 ms. The normalized collision energy was set to 35%
and 60% at an activation time of 20 ms and 50 ms for MS2 and MS3 scans,
respectively.
Database searches and data filtering and quantitation
Following mass spectrometric acquisition, the RAW files were converted into the
mzXML format and individual precursors selected for MS2 fragmentation were
checked for erroneous monoisotopic m/z and the mass measurements refined (4). The
MS/MS spectra were searched against the human ipi database (version 3.6) using the
Sequest algorithm (version 28) (5). A decoy database search approach was employed,
where each protein sequence was listed in both the forward and reversed direction, to
enable the estimation of the peptide and protein identification false discovery rates
3 Diabetes Page 32 of 61
(FDR). The search parameters were as follows: 50 ppm precursor ion tolerance; 1.0
Da product mass tolerance; variable modifications:oxidation of methionine
(+15.9949); fixed modifications: carbamidomethylation of cysteine (+57.0214), Six plex TMT tags on lysine residues and peptide N termini (+ 229.162932 Da). Two different types of searches were performed. Firstly, LysC specificity was required and secondly, 'no enzyme specificity' was required. On account of the large numbers of proteolytic enzymes known to be present in pancreatic fluid, it is likely that during the clinical sampling of pancreatic fluid degradation by enzymes had occurred in addition to the desired Lys C digestion, hence the reason to search with 'no enzyme' specified'.
The data was filtered to a false discovery rate of less than 1% based on the target decoy database approach (6). Filtering using linear discriminant analysis was performed to generate a classifier to distinguish between correct and incorrect identifications based on the following parameters: XCorr, Cn, peptide ion mass accuracy, charge state and peptide length, as described (4).
Further filtering of the list to include only 'pancreas specific' proteins was based on analyses of nine different mouse tissues (4)
(https://gygi.med.harvard.edu/phosphomouse/index.php).
To test the hypothesis that secretin stimulated duodenal secretory proteins truly reflected pancreatic disease, an investigator (F.M.) blinded to the samples, performed unsupervised clustering analysis based on protein abundance (i.e. algoritmic classification procedure to group samples based on protein expression similarities and blinded to a knowledge of sample disease status).
Peptide quantification using TMT reporter ion intensity was performed using in house software, as described previously (3). Briefly, a 0.06 m/z window around the theoretical m/z value of each reporter ion (126, 127, 128, 129, 130 and 131) was
4 Page 33 of 61 Diabetes
scanned for ions, and the intensity of the signal nearest to the theoretical m/z value
was recorded. The intensities of the reporter ion intensities were denormalised by
multiplying them with the ion accumulation time for each MS3 spectrum and.
Adjustment based on the overlap of isotopic envelopes for the reporter ions was
performed.
Immunoblotting. Secretions from the pancreas (15 microgram applied to each well;
concentration estimated by BCA assay; Pierce) was applied directly to and separated
by SDS PAGE, electrophoretically transferred to a nitrocellulose membrane and
stained with anti keratin 18 antibody (4548S ;1:2000; Cell Signaling Technology),
anti SKP1 antibody (2156S; 1:500; Cell Signaling Technology), anti PKM2 antibody
(4053S; 1:1000; Cell Signaling Technology), anti LMNA (4777S;1:1000; Cell
Signaling Technology), anti GSN (AB2969; 1:2000; Millipore) or anti amylase
(3796S;1:500; Cell Signaling Technology). Staining was developed using a
chemoilluminescence method (Pierce ECL Western blotting substrate; Pierce).
Kinase activity assay
The procedures were similar to that described (7) but with a few changes to the
method as described below. A set of 60 synthetic peptide substrates, each at 5 M,
were incubated with 10 g lysate and kinase reaction buffer containing Tris Cl (25
mM, pH 7.5), ATP (5 mM), MgCl2 (7.5 mM), EGTA (0.2 mM), β glycerophosphate
(7.5 mM), Na3VO4 (0.1 mM) and DTT (0.1 mM). After incubation for 45 minutes at
room temperature, the reaction was quenched with 100 L of 1% trifluoroacetic acid
(TFA). A known amount (5 pmol) of internal standard phosphorylated stable isotope
labeled peptides were spiked into the quenched reaction. The solution was desalted
5 Diabetes Page 34 of 61
using Sep Pak C18 50 mg cartridges (Waters) and dried using vacuum centrifugation.
Different to the previous method (7), enrichment for phosphoproteins using immobilized metal affinity chromatrography (IMAC) was not performed. The desalted sample were resuspended in 100 L of 5% formic acid of which 2 L was then subjected to analysis by reverse phase liquid chromatography mass spectrometry. Technical duplicates were performed for each lysate.
Kinase abbreviation
Category Kinase
AA PKA
CC PKC
DD Acidic
MM MAPK
PP Proline directed
RR RSK
KK AKT
XX pY_group_1
YY pY_group_2
ZZ pY_group_3
QQ DNA damage
GG GSK3
CH CHEK
OO Other S/T kinase
WW Y other
6 Page 35 of 61 Diabetes
BB Basic
AM AMPK
Proteolytic degradation assay
A set of 45 synthetic peptide substrates, each at 1 M, were incubated with 10 g
pancreatic fluid and reaction buffer containing Tris Cl (25 mM, pH 7.5), MgCl2 (7.5
mM), EGTA (0.2 mM), β glycerophosphate (7.5 mM), Na3VO4 (0.1 mM) and DTT
(0.1 mM). The reaction was incubated at room temperature for 0, 15, 60 min, after
which the reaction was quenched with 100 L of 1% trifluoroacetic acid (TFA).
Twenty five pmol of internal standard stable isotope labeled substrate peptides was
spiked into the quenched reaction. The solution was desalted using Sep Pak C18 50
mg cartridges (Waters) and dried using vacuum centrifugation. The desalted sample
was then resuspended in 100 L of 5% formic acid of which 2 L was then subjected
to analysis by mass spectrometry. Technical duplicates were performed for each
sample at each time point.
The peptides from the kinase activity and proteolytic degradation assays were
analysed by LC MS on a high resolution Exactive Orbitrap mass spectrometer
(Thermo). The experimental details are outlined in the Suppl. information. Briefly, a
45 minute gradient was used to separate the peptides from 10 37% solvent B (0.125%
formic acid in acentonitrile) at a flow rate of 300 mL/min. LC MS data was collected
from 350 1500 m/z and the extracted ion chromatograms of the light and heavy
peptides were used to quantitate the absolute kinase activity and the extent of peptide
degradation respectively. Data analysis was performed using Pinpoint (Thermo).
7 Diabetes Page 36 of 61
Mass spectrometry and data analysis for kinase and proteolytic degradation assays
The peptides from the kinase activity and proteolytic degradation assay, dissolved in
5% formic acid, were analysed by LC MS on a high resolution Exactive Orbitrap mass spectrometer (Thermo) equipped with a Nanospray II electrospray ionisation source (Thermo) coupled to a Thermo binary pump and a Famos autosampler (LC
Packings). Peptides were separated on a hand pulled fused silica microcapillary with an internal diameter of 175 m, 15 cm long, packed with a C18 reversed phase resin
(Magic C18AQ, particle size 5 m, pore size 200 Å, Michrom Bioresources, Auburn,
CA). The needle tip had an internal diameter of approximately 5 m. Once loaded, the peptides were separated across a 45 minute linear gradient of 10 37% solvent B
(0.125 % FA in ACN) at a flow rate of 300 nl/min provided across a flow splitter by the HPLC pumps. Buffer A comprised 0.125% FA and 3% ACN in water. Data was collected from 350 1500 m/z full MS with a resolution setting of 60,000.
Analysis was performed using the commercial software package Pinpoint
(Thermo).The amount of substrate phosphorylation was determined by comparing the area of the light phosphorylated substrate peptide with that of the heavy phosphorylated internal standard peptide. The phosphorylated substrate peptides are chemically identical to the heavy internal standard and therefore should have the same chromatographic characteristics and ionisation efficiency. In the case of the proteolytic degradation assay, the amount of peptide degradation was determined by comparing the area of the light substrate (degraded during the reaction) with that of
8 Page 37 of 61 Diabetes
the heavy substrate internal standard peptide that was added following quenching of
the reaction. Extracted ion chromatograms were generated from the MS scan with ±
10 ppm mass accuracy around the monoisotopic peak of the most abundant charge
state. The extracted ion chromatograms were integrated using Pinpoint. Whilst
Pinpoint generates the area of the extracted ion chromatograms semi automatically,
each chromatogram required manual inspection to ensure that the correct boundaries
had been selected. Where this was not the case, the peak boundaries were manually
altered. It was required that heavy and light peptides co eluted perfectly for accurate
quantitation. Measurements were excluded where the peak height was less than 10000
or where the peak area was less than 1% of the internal standard (50 fmol). Since a
known amount of internal standard was added to the reaction (5 pmol for the kinase
assay, 25 pmol for the proteolytic degradation assay) prior to purification, the
absolute amount of product formed could be determined. The product formed/kinase
activity is presented with units of fmol/ g/min. The ratio of the light to heavy peptide
area was divided by the amount of lysate (10 g) and the time of the reaction (45 min
for the kinase activity assay). For the proteolytic digestion assay, the degradation is
normalized to the light peptide/heavy peptide ratio for the zero time point.
References
1. Ræder H, Johansson S, Holm PI, Haldorsen IS, Mas E, Sbarra V, Nermoen I, Eide SA, Grevle L, Bjørkhaug L, Sagen JV, Aksnes L, Søvik O, Lombardo D, Molven A, Njølstad PR: Mutations in the CEL VNTR cause a syndrome of diabetes and pancreatic exocrine dysfunction. Nat Genet 2006;38:54 62 2. Villen J, Gygi SP: The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry. Nature protocols 2008;3:1630 1638 3. Ting L, Rad R, Gygi SP, Haas W: MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics. Nature methods 2011;
9 Diabetes Page 38 of 61
4. Huttlin EL, Jedrychowski MP, Elias JE, Goswami T, Rad R, Beausoleil SA, Villen J, Haas W, Sowa ME, Gygi SP: A tissue specific atlas of mouse protein phosphorylation and expression. Cell 2010;143:1174 1189 5. Eng JK, McCormack AL, Yates JR, 3rd: An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom. 1994;5:976 989 6. Elias JE, Gygi SP: Target decoy search strategy for increased confidence in large scale protein identifications by mass spectrometry. Nature methods 2007;4:207 214 7. Kubota K, Anjum R, Yu Y, Kunz RC, Andersen JN, Kraus M, Keilhack H, Nagashima K, Krauss S, Paweletz C, Hendrickson RC, Feldman AS, Wu CL, Rush J, Villen J, Gygi SP: Sensitive multiplexed analysis of kinase activities and activity based kinase identification. Nat Biotechnol 2009;27:933 940
Supplementary Figure Legends and Supplementary Table Legends
Suppl. Fig. 1: Protein-protein interaction modeling of upregulated pancreatic juice proteins in CEL mutation carriers. Using Cytoscape, the figure shows major protein protein interactions among the proteins identified in pancreatic juice based on Gene
Network Enrichment Analysis. Note the direct interaction between YWHAZ and
KRT18, two proteins that were identified in the pancreatic juice protein analyses.
More generally, the immediate set of protein protein interactors to the YWHAZ encoded protein, comprising the proteins encoded by YWHAE, ATP5A1, EPB41L3,
PPP1CC, ENO1, and KRT18 was together highly significant (Fisher’s p value =
0.00028).
Suppl. Table 1: Clinical, imaging and cytokine characteristics for CEL mutation carriers and controls.
Suppl. table 2: Protein expression values for proteins identified in the pancreatic proteome by proteomics analysis. Log2 ratio, Log2 of the ratio of mass spectrometry
10 Page 39 of 61 Diabetes
abundance for the sum of the three healthy controls divided by the sum of the three
diabetic mutation carriers. Log 2 total, Log 2 of the total mass spectrometry
abundance for a protein as the sum of the abundances of three healthy controls and the
three diabetic mutation carriers.
Suppl. table 3: PubMed IDs documenting the literature support of pathways
involved in the MAPK-targeted proteins based on the output from the Biobase
Explain tool.
Supplementary Figure and Tables
Suppl. Fig. 1
11 Diabetes Page 40 of 61
Suppl Table 1:
Status Control Control Control Control Control Control Control Control CEL CEL (N2) (N1) (N3) carrier carrier Sex F F M F F F M M M M Age (yrs) 32 38 40 50 53 64 63 25 9 14 EGF 442.88 110.76 20.13 39.72 110.37 63.48 143.63 128.28 101.22 32.19 EOTAXIN 0 1.55 1.07 0 0 1.58 1.79 7.26 1.18 0.53 FGF-2 0 2.5 0 0 0 8.66 2.50 16.71 2.5 0 Flt-3 1.03 0 0 0 0 3.85 2.49 7.86 1.77 3.18 ligand fractalkine 0 0 0 0 0 0 <1.28 <1.28 0 0 G-CSF 1.11 0 0 0 0 0 <0.42 0.59 0 1.11 GM-CSF 0 0.72 0.96 0 0 0.82 0.74 1.49 2.17 0.74 GRO 2.46 7.32 5.91 0 2.46 9.73 4.32 11.29 2.46 11.05 IFN-a2 0 0 0 0 0 0 <1.05 <1.05 0 0 IFN-G 0 0 0 0 0 1.68 <1.38 2.92 0 0 IL-1a 2.08 0.56 20.53 2.32 0.68 16.15 510.68 79.36 35.79 5.48 IL-1b 0 0 0 0 0 0.76 8.71 1.97 1.14 3.67 IL-1ra 0.79 1.77 7.37 1.91 0 3.51 67.50 10.07 6.38 13.15 IL-2 0 0 0.45 0 0 0.45 <0.45 2.06 0 0 IL-3 4 16.28 4.94 11.56 0 8.52 6.26 12.45 0 0 IL-4 0 0 0 0 0 0 <0.84 <0.84 0 0 IL-5 0 0 0 0 0 0 0.18 <0.16 0 0.2 IL-6 0 0 0 0 0 0.52 1.20 5.19 0 4.75 IL-7 0 0 0 0 0 0 <0.62 <0.62 0 3.56 IL-8 0.31 0 1.52 0.22 0.14 2.13 2.64 4.40 1.95 4.53 IL-9 0 0 0 0 0 0 <1.12 <1.12 0 0 IL-10 0 0 0 0.8 0 0.39 6.40 2.25 0 9.45 IL- 0 0.9 0 1.04 0 3.21 5.02 10.94 6.13 7.25 12(P40) IL- 0 0 1.28 0 0 0 <1.23 3.93 0 0 12(P70) IL-13 0 0 0 0 0 0 2.81 2.02 0 3.47 IL-15 0 0 1.99 0 1.85 2.84 2.70 5.41 0 1.65 IL-17A 0 0 0 0 0 0 <0.36 0.90 0 0 IP-10 0 0 0 0 0 0 <0.41 7.08 0 0 MCP-1 16.4 1.07 1.07 0 0 1.07 <0.38 1.40 104.43 23.22 MCP-3 3.64 3.64 4.49 0 0 8.15 4.49 12.96 0 7.49 MDC 3.25 2.32 7.74 0 0 10.24 <1.98 14.83 0 13.36 MIP-1a 0 1.73 7.1 0 0 15.78 14.93 22.33 0 18.64 MIP-1b 0 0 0 0 0 0 <0.41 4.40 0 0
12 Page 41 of 61 Diabetes
PDGF-AA 75.46 69.6 64.7 73.02 135.13 118.56 149.74 73.18 58.35 109.98 PDGF- 0 0 0 0 0 0 19.27 10.71 0 0 AB/BB RANTES 17.26 0 0 0 0 0 <1.51 <1.51 1.77 7.38 sCD-40L 1.56 0.99 0.99 0.51 0.99 1.88 0.51 1.41 1.26 2.21 sIL-2Ra 0 1.9 0 0 0 2.12 <0.7 3.44 0 0 TGF-a 1.4 0 0 0.63 0.7 0 <0.82 1.28 0 0 TNF-a 0 0 0 0 0 0 <0.42 <0.42 0 0 TNF-b 0 0 0 0 0 0 <0.6 <0.6 0 1.41 VEGF 5.55 0 5.55 0 0 12.51 <0.88 16.84 2.02 12.51
Status CEL CEL CEL CEL CEL CEL CEL CEL CEL CEL carrier carrier carrier carrier carrier carrier carrier carrier carrier carrier (P2) (P1) (D2) (D4) (D1) (D3) (C1) Sex M M F F M M F M M F Age (yrs) 37 40 43 45 56 57 57 65 69 78 EGF NA 44.74 80.68 23.95 5.5 13.84 125.19 1.76 6.85 19.4 EOTAXIN NA 1.07 0.92 0.1 68.8 0.41 0.57 3.24 40.57 0.71 FGF-2 NA 3.9 19.62 0 14.3 7.57 10.2 3.9 12.57 13.45 Flt-3 NA 0 6.26 0 6.81 3.18 1.77 1.03 5.69 1.77 ligand fractalkine NA 0 0 0 26.05 0 0 0 487 0 G-CSF NA 0 0.59 0 26.4 3.86 0 0 0 1.95 GM-CSF NA 0.35 1.73 0.19 1.33 0.77 0.77 1.54 2.98 1.58 GRO NA 7.32 116.7 45.11 97.31 15.2 109.7 2.46 88.81 161.02 IFN-a2 NA 0 7.32 0 2.1 0 0 0 0 3.9 IFN-G NA 0 0 0 0 0 0 0 1.45 6.45 IL-1a NA 33.67 273.25 109.59 14.57 37.1 594.34 10.72 6.5 2.16 IL-1b NA 2.39 2.82 1.5 0.67 0 1.24 0 0.76 0 IL-1ra NA 16.26 22.85 186.32 69.2 150.09 41.18 7.71 43.41 15.74 IL-2 NA 0.5 0.54 0 0.74 0.74 0.54 0.45 1.22 0.74 IL-3 NA 0 0 0 0 0 0 5.64 0 0 IL-4 NA 0 2.1 0 3.3 0 0 0 0 0 IL-5 NA 0 0.28 0 0 0 0 0 0 0 IL-6 NA 0 5.19 0 0 0 0 0 0 15.46 IL-7 NA 0 1.36 0 6.23 0 0 0 0 0 IL-8 NA 7.67 21.1 16.76 4.28 3.08 373.59 1.16 4.4 167.74 IL-9 NA 0 0 0 0 0 0 0 0 0 IL-10 NA 0.2 1.53 0.86 0.5 0.39 0 0 0.2 0 IL- NA 2.16 18.45 0 4.29 1.83 1.5 7.8 5.02 0 12(P40) IL- NA 0 1.44 0 0 0 0 0 0 0 12(P70)
13 Diabetes Page 42 of 61
IL-13 NA 0 2.33 0 11.32 0 0 0 0 0 IL-15 NA 1.85 5.96 0 3.14 3.28 0 4.49 3.21 0 IL-17A NA 0 0 0 0 0 0 0.39 1.47 0 IP-10 NA 0 50.3 0 333.95 23.96 13.9 3.89 165.19 1.12 MCP-1 NA 0 119.07 10.28 225.49 48.74 34.65 5.89 441.82 61.38 MCP-3 NA 6.79 14.62 3.64 6.79 7.49 5.3 3.64 11.14 6.43 MDC NA 4.18 24.01 5.09 16.26 28.57 6.87 3.25 21.56 3.25 MIP-1a NA 4.31 22.97 0 18.39 0 0 10.42 26.41 3.6 MIP-1b NA 0 14.68 0 19.84 0 0 0 39.28 9.49 PDGF-AA NA 55.77 202.14 53.72 61.89 61.12 51.57 370.6 74.34 23.48 PDGF- NA 0 0 0 0 0 0 0 0 0 AB/BB RANTES NA 0 31.26 35.13 202.69 106.27 10.65 0 69.44 4.71 sCD-40L NA 1.88 2.92 0.73 4.47 2.21 0.99 0.73 4.07 2.56 sIL-2Ra NA 0 0.74 0 10.45 2.01 0 0 4.35 1.2 TGF-a NA 0 12.11 0 8.63 0 8.27 0 3.16 4.96 TNF-a NA 0 0 0 0.45 0 0 0 0 0 TNF-b NA 0 0.73 0 0 0 0 0 0 0 VEGF NA 0 50.92 0 176.64 6.68 0 0 21.28 32.91
Suppl Table 2:
Gene D1 D3 D2 N2 N3 N1 CLEC3B 1.0E+06 5.5E+06 3.1E+06 5.0E+01 5.0E+01 5.0E+01 ATP5A1 1.1E+06 2.9E+06 4.4E+06 5.0E+01 5.0E+01 5.0E+01 KRT18 1.5E+06 1.2E+06 3.0E+06 5.0E+01 5.0E+01 5.0E+01 CAPZA1 7.4E+05 1.4E+06 2.8E+06 5.0E+01 5.0E+01 5.0E+01 LOC100133511 7.3E+05 1.3E+06 2.4E+06 5.0E+01 5.0E+01 5.0E+01 FETUB 1.2E+05 1.7E+06 2.5E+06 5.0E+01 5.0E+01 5.0E+01 HSPD1 5.0E+01 7.5E+05 3.5E+06 5.0E+01 5.0E+01 5.0E+01 GPR126 1.5E+05 2.5E+06 5.8E+05 5.0E+01 5.0E+01 5.0E+01 TPM1 7.7E+04 5.7E+05 1.5E+06 2.0E+01 5.0E+01 5.0E+01 SUCLG2 2.7E+05 9.6E+05 1.9E+06 5.0E+01 1.1E+02 5.0E+01 TTBK2 8.8E+04 2.8E+05 1.9E+06 5.0E+01 5.0E+01 5.0E+01 SERPINA10 5.0E+01 1.4E+06 7.0E+05 5.0E+01 5.0E+01 5.0E+01 ATP1A2 5.0E+01 4.1E+05 2.6E+06 5.0E+01 1.5E+02 5.0E+01 LUM 5.0E+01 1.2E+06 6.2E+05 5.0E+01 5.0E+01 5.0E+01 BCAS1 6.9E+04 6.9E+05 1.1E+06 5.0E+01 5.0E+01 5.0E+01 HPGD 5.0E+01 4.0E+05 1.4E+06 5.0E+01 5.0E+01 5.0E+01
14 Page 43 of 61 Diabetes
ATP6V1E1 4.2E+05 4.9E+05 8.8E+05 5.0E+01 5.0E+01 5.0E+01 AKR1D1 2.6E+05 1.1E+06 3.5E+05 5.0E+01 5.0E+01 5.0E+01 MUPCDH 5.0E+01 4.4E+05 1.2E+06 5.0E+01 5.0E+01 5.0E+01 EFNA1 5.0E+01 1.1E+06 5.3E+05 5.0E+01 5.0E+01 5.0E+01 CYB5A 5.5E+05 4.3E+05 6.2E+05 5.0E+01 5.0E+01 5.0E+01 CRNN 3.3E+05 6.1E+05 6.3E+05 5.0E+01 5.0E+01 5.0E+01 TRAP1 7.2E+04 5.6E+05 8.3E+05 5.0E+01 5.0E+01 5.0E+01 IGLC1;IGLV1- 4.2E+06 7.2E+05 5.0E+01 4.5E+02 5.0E+01 5.0E+01 44;IGLV1-40;IGLV3- 21;IGLV2-11;IGLV2- 14;IGL@;IGLC2;IGLC 3 APOL1 5.0E+01 1.2E+06 9.0E+04 5.0E+01 5.0E+01 5.0E+01 HIBADH 2.9E+05 3.2E+05 7.0E+05 5.0E+01 5.0E+01 5.0E+01 COL1A1 2.0E+05 7.7E+05 2.7E+05 5.0E+01 5.0E+01 5.0E+01 IPI00386135 1.7E+06 5.0E+01 4.1E+03 1.3E+02 5.0E+01 5.0E+01 AK3 2.6E+05 3.6E+05 5.1E+05 5.0E+01 5.0E+01 5.0E+01 ARHGDIA 2.3E+05 2.9E+05 5.8E+05 5.0E+01 5.0E+01 5.0E+01 ATP5B 1.0E+06 1.4E+06 1.4E+06 5.0E+01 4.4E+02 5.0E+01 EPS8 1.1E+05 4.0E+05 5.5E+05 5.0E+01 5.0E+01 5.0E+01 COPZ1 1.9E+05 1.9E+05 6.8E+05 5.0E+01 5.0E+01 5.0E+01 PSME1 1.8E+05 4.6E+05 4.1E+05 5.0E+01 5.0E+01 5.0E+01 PTGR1 1.1E+05 4.0E+05 5.1E+05 5.0E+01 5.0E+01 5.0E+01 DSC2 1.7E+05 2.3E+05 1.0E+06 5.0E+01 1.3E+02 5.0E+01 SIRPA 1.2E+05 8.2E+05 6.2E+02 5.0E+01 5.0E+01 5.0E+01 ARPC5 1.6E+05 3.8E+05 3.7E+05 5.0E+01 5.0E+01 5.0E+01 NUCB2 2.2E+05 1.5E+05 4.8E+05 5.0E+01 5.0E+01 5.0E+01 PBLD 5.0E+01 2.3E+05 6.2E+05 5.0E+01 5.0E+01 5.0E+01 SULT1A3;SULT1A4 5.0E+01 2.6E+05 5.7E+05 5.0E+01 5.0E+01 5.0E+01 APOF 5.0E+01 5.7E+05 2.4E+05 5.0E+01 5.0E+01 5.0E+01 HEPH 1.8E+05 1.3E+05 3.7E+05 5.0E+01 5.0E+01 5.0E+01 HSPA9 3.8E+05 2.7E+05 1.9E+03 5.0E+01 5.0E+01 5.0E+01 DDX3X 9.9E+04 1.1E+05 4.2E+05 5.0E+01 5.0E+01 5.0E+01 RPL17;LOC1001339 5.0E+01 1.6E+05 4.6E+05 5.0E+01 5.0E+01 5.0E+01 31 DGAT1 1.3E+05 2.3E+05 2.5E+05 5.0E+01 5.0E+01 5.0E+01 COPB1 5.0E+01 1.9E+05 4.1E+05 5.0E+01 5.0E+01 5.0E+01 PGLYRP2 6.4E+04 3.4E+05 1.9E+05 5.0E+01 5.0E+01 5.0E+01 ARPC2 5.0E+01 1.9E+05 3.6E+05 5.0E+01 5.0E+01 5.0E+01 DBI 1.8E+05 2.1E+05 1.4E+05 5.0E+01 5.0E+01 5.0E+01 IGHG1;IGHG2;IGHV4 5.2E+05 5.0E+01 6.6E+04 5.0E+01 7.4E+01 5.0E+01 -31;IGH@ MYH14 2.9E+05 2.3E+05 6.4E+05 5.0E+01 2.7E+02 5.0E+01 IGLV10-54 1.6E+05 3.1E+05 8.1E+02 5.0E+01 5.0E+01 5.0E+01 DCI 2.4E+05 2.2E+05 3.3E+05 5.0E+01 1.7E+02 5.0E+01
15 Diabetes Page 44 of 61
MYL1 9.6E+04 1.7E+05 1.7E+05 5.0E+01 5.0E+01 5.0E+01 KIAA1967 5.0E+01 1.5E+05 2.7E+05 5.0E+01 5.0E+01 5.0E+01 IPI00384394 4.2E+05 5.0E+01 2.1E+03 5.0E+01 5.0E+01 5.0E+01 SLC16A1 5.0E+01 1.1E+05 3.1E+05 5.0E+01 5.0E+01 5.0E+01 UGT2B28 4.3E+05 5.0E+01 1.2E+05 5.0E+01 1.4E+02 5.0E+01 ARPC4;TTLL3 5.0E+01 1.0E+05 2.4E+05 5.0E+01 5.0E+01 5.0E+01 RPLP2 7.9E+05 2.2E+06 6.4E+06 4.1E+03 5.0E+01 5.0E+01 SKP1 7.0E+04 1.3E+05 7.4E+04 5.0E+01 5.0E+01 5.0E+01 RPLP0 3.7E+05 1.2E+06 1.9E+06 2.1E+03 5.0E+01 5.0E+01 PSMD2 5.0E+01 1.2E+05 1.2E+05 5.0E+01 5.0E+01 5.0E+01 ACAA1 5.0E+01 8.4E+04 2.8E+05 5.0E+01 1.9E+02 5.0E+01 PHB 5.5E+05 6.8E+05 1.2E+06 5.0E+01 5.0E+01 1.9E+03 IPI00017870 1.7E+06 1.8E+06 5.0E+06 7.1E+03 5.0E+01 5.0E+01 LIPF 1.8E+05 5.0E+01 2.9E+05 5.0E+01 3.4E+02 5.0E+01 RAB14 1.3E+05 5.0E+01 2.8E+05 5.0E+01 3.3E+02 5.0E+01 HNRNPK 1.2E+05 5.0E+01 1.8E+05 5.0E+01 2.1E+02 5.0E+01 RPN1 1.2E+05 5.0E+01 2.0E+05 5.0E+01 2.4E+02 5.0E+01 MARCKSL1 5.0E+01 5.6E+04 8.1E+04 5.0E+01 5.0E+01 5.0E+01 DDAH1 9.9E+04 5.0E+01 1.9E+05 5.0E+01 2.2E+02 5.0E+01 SPTAN1 8.9E+04 5.0E+01 9.4E+04 5.0E+01 1.1E+02 5.0E+01 SPRR2C;SPRR2G 1.3E+05 5.0E+01 6.3E+02 5.0E+01 5.0E+01 5.0E+01 RPLP1 5.0E+01 5.0E+01 2.8E+06 5.0E+01 3.3E+03 5.0E+01 ALPI 7.0E+04 5.0E+01 2.3E+05 5.0E+01 2.7E+02 5.0E+01 AK2 2.0E+06 2.1E+06 1.9E+06 5.0E+01 5.0E+01 7.6E+03 ALDH3A2 6.4E+04 5.0E+01 1.2E+05 5.0E+01 1.4E+02 5.0E+01 IGLV3-25 5.0E+01 5.0E+01 5.7E+05 5.0E+01 6.7E+02 5.0E+01 SFN 5.0E+01 5.0E+01 5.5E+05 5.0E+01 6.5E+02 5.0E+01 ICAM1 5.0E+01 1.0E+05 5.0E+01 5.0E+01 5.0E+01 5.0E+01 ELANE 5.0E+01 5.0E+01 3.0E+05 5.0E+01 3.5E+02 5.0E+01 EPB41L3 8.0E+05 1.4E+06 2.4E+06 5.0E+01 5.0E+01 6.9E+03 LOC723972;ANP32A 5.0E+01 9.6E+04 5.0E+01 5.0E+01 5.0E+01 5.0E+01 TXNDC17 5.0E+01 5.0E+01 2.6E+05 5.0E+01 3.0E+02 5.0E+01 PGA3;PGA4;PGA5 5.0E+01 5.0E+01 2.5E+05 5.0E+01 3.0E+02 5.0E+01 ITIH2 3.1E+06 1.5E+07 2.0E+07 3.4E+03 7.3E+02 5.7E+04 GPD1 5.0E+01 5.0E+01 1.7E+05 5.0E+01 2.0E+02 5.0E+01 UBE2V1;TMEM189- 5.0E+01 5.0E+01 1.6E+05 5.0E+01 1.8E+02 5.0E+01 UBE2V1 SERPINF1 1.4E+06 4.0E+06 3.8E+06 5.0E+01 5.0E+01 1.7E+04 GSTT1 5.0E+01 7.9E+04 5.0E+01 5.0E+01 5.0E+01 5.0E+01 MEP1B 5.0E+01 5.0E+01 1.0E+05 5.0E+01 1.2E+02 5.0E+01 CHGA 5.0E+01 7.0E+04 5.0E+01 5.0E+01 5.0E+01 5.0E+01 RANP1;RAN 5.0E+01 6.6E+04 5.0E+01 5.0E+01 5.0E+01 5.0E+01 YWHAH 5.0E+01 5.0E+01 8.3E+04 5.0E+01 9.8E+01 5.0E+01 YWHAQ 5.0E+01 5.0E+01 7.2E+04 5.0E+01 8.5E+01 5.0E+01 HIST2H2BE 3.6E+05 8.5E+04 1.7E+06 4.8E+03 4.9E+02 5.0E+01
16 Page 45 of 61 Diabetes
C9 1.3E+06 1.2E+07 5.7E+06 1.2E+04 5.0E+01 4.6E+04 HSP90AA1 1.1E+06 1.7E+06 2.2E+06 5.0E+01 5.0E+01 2.0E+04 APOC1 3.9E+05 4.6E+06 2.6E+06 5.0E+01 5.0E+01 3.0E+04 AGR2 8.5E+05 7.5E+05 1.1E+06 1.1E+04 5.0E+01 5.0E+01 AHSG 1.1E+07 2.6E+07 2.0E+07 1.0E+04 1.0E+05 1.2E+05 EIF5A2 2.3E+05 2.7E+06 5.2E+06 5.0E+01 5.0E+01 3.4E+04 FBP1 9.0E+05 1.9E+06 2.1E+06 5.0E+01 5.0E+01 2.5E+04 YWHAZ 6.8E+05 4.1E+05 5.7E+05 6.6E+01 6.6E+02 8.3E+03 PKM2 4.5E+05 2.3E+06 2.4E+06 5.0E+01 2.9E+04 5.0E+01 HIST1H2AB;HIST1H 1.8E+05 4.4E+05 5.2E+06 5.0E+01 5.0E+01 3.5E+04 2AE;HIST1H2AL;HIS T1H2AG;HIST1H2AJ; HIST1H2AK;HIST1H 2AI;HIST1H2AM HIST1H4J;HIST1H4E 4.5E+06 2.3E+06 9.7E+06 5.8E+02 2.4E+03 1.1E+05 ;HIST1H4H;HIST1H 4K;HIST2H4A;HIST1 H4D;HIST1H4F;HIST 2H4B;HIST1H4C;HIS T1H4B;HIST1H4I;HI ST1H4L;HIST1H4A; HIST4H4 AFM 1.7E+06 5.3E+06 3.7E+06 5.0E+01 5.0E+01 7.8E+04 IDH1 1.5E+06 2.3E+06 2.2E+06 5.0E+01 5.0E+01 4.5E+04 ETFB 1.1E+06 1.8E+06 2.3E+06 4.1E+03 5.0E+01 4.2E+04 PFN1 3.4E+06 3.1E+06 7.4E+06 5.0E+01 5.0E+01 1.3E+05 MYL6;MYL6B 5.0E+01 2.9E+05 1.1E+06 1.3E+04 5.0E+01 5.0E+01 CFL1 3.2E+05 2.1E+06 3.4E+06 5.0E+01 2.2E+02 5.5E+04 FABP2 4.4E+05 5.0E+01 7.3E+04 5.0E+01 9.0E+01 4.7E+03 APOA1 1.1E+05 7.6E+07 5.1E+07 5.2E+05 3.5E+05 4.0E+05 KRT19 6.0E+05 1.0E+06 3.2E+06 1.4E+04 1.3E+02 4.1E+04 CMAS 2.1E+05 2.1E+05 8.9E+05 5.0E+01 5.0E+01 1.7E+04 MYH14 3.8E+05 6.7E+05 4.0E+06 5.0E+01 9.2E+02 6.6E+04 PURA 9.4E+04 5.0E+01 1.6E+06 5.0E+01 1.9E+03 2.1E+04 HGFAC 6.5E+04 3.0E+06 1.0E+06 5.0E+01 5.6E+04 5.0E+01 ACSL5 2.5E+05 6.5E+05 7.1E+05 5.0E+01 2.6E+02 2.2E+04 IPI00398129 2.2E+05 5.8E+05 1.2E+06 2.7E+03 5.0E+01 2.7E+04 HIST2H3D;HIST2H3 6.6E+05 9.4E+04 4.9E+06 2.3E+04 1.3E+02 6.0E+04 C;HIST2H3A BTD 1.5E+06 5.9E+06 5.0E+06 5.0E+01 1.0E+04 1.7E+05 UQCRC2 5.7E+06 4.0E+06 9.8E+06 5.0E+01 5.0E+01 2.9E+05 CPS1 8.1E+05 3.7E+06 4.6E+06 1.0E+02 3.8E+04 1.0E+05 ANXA13 2.3E+05 2.1E+06 6.5E+06 5.0E+01 1.2E+05 3.1E+04 HNRNPL 1.3E+06 5.8E+05 1.5E+06 1.3E+04 5.0E+01 4.7E+04 PROC 5.9E+04 1.2E+06 6.3E+05 5.0E+01 5.0E+01 3.4E+04 APOA4 9.0E+05 7.0E+07 3.5E+07 1.2E+06 4.0E+05 4.4E+05
17 Diabetes Page 46 of 61
TST 2.0E+05 2.5E+05 1.3E+06 5.0E+01 6.2E+02 3.4E+04 HRG 6.1E+06 9.4E+06 6.8E+06 4.5E+04 2.8E+05 1.3E+05 NP 1.7E+06 1.6E+06 3.1E+06 1.1E+05 5.0E+01 2.0E+04 LMNA 7.2E+05 5.1E+05 1.2E+06 9.6E+03 5.0E+01 4.3E+04 DEFA5 3.8E+06 3.2E+06 7.0E+06 1.2E+02 1.4E+05 1.7E+05 CNDP1 2.3E+05 1.7E+06 9.5E+05 5.0E+01 2.9E+04 4.6E+04 MYH9 1.8E+06 3.7E+06 1.6E+07 4.5E+05 7.1E+04 4.3E+04 SLC9A3R1 2.7E+06 4.4E+06 3.4E+06 2.0E+04 1.5E+05 1.2E+05 HYOU1 1.2E+06 1.4E+06 1.6E+06 5.0E+01 5.0E+01 1.2E+05 HBG1;HBG2 2.2E+05 8.1E+06 3.6E+06 5.0E+01 1.0E+05 2.4E+05 PRDX5 9.6E+05 1.2E+06 1.2E+06 2.0E+04 5.0E+01 7.8E+04 KRT8 5.8E+06 5.5E+06 1.9E+07 1.3E+05 3.0E+05 4.4E+05 EEF1A1 5.0E+01 5.0E+05 1.5E+06 5.9E+04 5.0E+01 5.0E+01 ACO2 1.2E+06 1.3E+06 2.3E+06 5.0E+01 3.1E+02 1.4E+05 GKN1 4.7E+04 1.4E+02 4.0E+06 8.6E+04 3.5E+04 5.0E+01 HSPA7 6.3E+05 7.6E+05 1.2E+06 5.0E+01 5.0E+01 7.9E+04 AKR7A3 3.0E+05 1.1E+06 1.1E+06 5.3E+04 5.0E+01 2.3E+04 APOC3 5.0E+01 1.4E+06 6.6E+05 1.0E+04 5.0E+01 5.3E+04 REG4 1.7E+05 7.9E+05 2.8E+06 6.5E+04 5.7E+04 5.0E+01 ACAT1 2.2E+06 2.1E+06 4.5E+06 5.0E+01 7.9E+04 2.0E+05 UQCRB 7.1E+05 6.0E+05 9.7E+05 5.0E+01 5.0E+01 7.5E+04 ITIH3 8.8E+05 2.8E+06 3.2E+06 1.8E+02 5.0E+01 2.3E+05 GLUD1 2.6E+06 1.5E+06 1.7E+06 5.0E+01 5.0E+01 2.0E+05 PGC 1.4E+07 1.5E+06 1.0E+07 2.0E+05 4.0E+05 2.8E+05 AGT 3.0E+06 9.5E+06 5.9E+06 1.5E+04 9.5E+01 6.4E+05 CKMT1B;LOC10013 5.0E+01 1.2E+06 2.1E+06 6.0E+04 5.0E+01 5.7E+04 3623;CKMT1A APOA2 6.0E+04 6.5E+06 3.6E+06 2.8E+04 5.1E+04 2.9E+05 IPI00382440 6.7E+05 8.4E+05 1.2E+06 5.0E+01 5.0E+01 1.0E+05 PCDH24 2.0E+05 2.8E+06 5.0E+05 5.0E+01 1.3E+05 5.0E+01 PDIA3 6.4E+06 4.0E+06 2.5E+06 2.6E+04 5.0E+01 4.8E+05 C8B 2.7E+05 1.9E+06 6.3E+05 5.0E+01 1.1E+05 5.0E+01 CFI 5.7E+06 1.9E+07 1.0E+07 1.2E+05 7.6E+05 5.2E+05 IGFALS 3.2E+05 5.2E+05 4.1E+05 5.0E+01 5.2E+04 5.0E+01 ARCN1 8.6E+05 1.1E+06 1.0E+06 7.6E+04 5.0E+01 5.9E+04 PRDX3 1.4E+06 1.0E+06 2.2E+06 1.6E+03 6.3E+04 1.5E+05 IPI00382489 2.9E+06 7.8E+05 2.8E+05 5.0E+01 4.9E+04 1.3E+05 VNN1 1.7E+05 9.4E+05 7.6E+05 5.0E+01 8.8E+04 5.0E+01 SLC5A1 2.5E+05 3.8E+05 3.5E+05 5.0E+01 5.0E+01 4.7E+04 ETFA 2.9E+06 2.9E+06 1.0E+06 8.5E+03 6.2E+04 2.6E+05 HSP90B1 2.9E+06 3.2E+06 3.7E+06 5.0E+01 2.5E+05 2.2E+05 CD59 6.8E+06 6.5E+06 3.3E+06 5.3E+03 2.9E+05 5.3E+05 IPI00782983 6.9E+05 1.1E+06 8.6E+04 5.0E+01 5.0E+01 9.2E+04 OAT 1.1E+06 1.5E+06 2.7E+06 5.0E+01 1.8E+05 8.6E+04 PCK2 7.5E+05 7.8E+05 7.9E+05 5.0E+01 6.8E+04 5.2E+04
18 Page 47 of 61 Diabetes
IGJ 3.5E+07 1.0E+07 3.3E+07 2.4E+05 1.9E+06 2.1E+06 MYO1A 2.9E+05 1.9E+05 1.6E+05 5.0E+01 5.0E+01 3.5E+04 LCT 2.8E+05 7.3E+05 2.4E+06 1.5E+04 8.0E+04 9.6E+04 UQCRC1 4.7E+05 7.6E+05 1.8E+06 5.0E+01 1.1E+05 6.4E+04 RAB35 3.7E+05 2.2E+05 8.3E+05 8.1E+04 2.8E+02 5.0E+01 IPI00829624 9.5E+05 3.3E+05 2.3E+05 5.0E+01 5.0E+01 8.6E+04 B2M 6.0E+05 1.4E+05 1.1E+05 3.4E+04 5.0E+01 1.5E+04 KRT4 2.6E+06 1.6E+06 8.9E+06 2.8E+05 3.7E+05 1.3E+05 PPA1 1.5E+06 9.2E+05 1.9E+06 1.4E+05 1.1E+03 1.2E+05 CPN2 1.7E+05 5.0E+05 2.3E+05 5.0E+01 1.3E+02 5.4E+04 IPI00003469 6.8E+05 7.7E+05 5.0E+01 5.0E+01 3.8E+04 5.1E+04 PRB3 4.4E+05 3.1E+02 1.4E+06 8.7E+04 6.1E+02 2.5E+04 NPC2 4.2E+05 2.7E+05 4.8E+05 1.9E+04 5.0E+01 5.5E+04 KNG1 9.8E+06 2.9E+07 2.1E+07 3.2E+05 1.7E+06 1.8E+06 PRSS8 2.3E+05 3.7E+05 4.9E+05 5.1E+02 3.9E+04 3.1E+04 EEF2 1.4E+06 3.0E+06 5.9E+06 3.3E+05 1.9E+05 1.5E+05 ITIH1 9.6E+06 1.6E+07 1.8E+07 5.5E+03 2.6E+05 2.7E+06 ACTN2 7.0E+05 1.1E+02 4.4E+05 2.6E+04 5.0E+01 5.3E+04 PGA3;PGA4;PGA5 1.2E+07 2.5E+06 2.7E+07 1.9E+06 2.3E+05 8.7E+05 ALDOB 1.3E+07 1.4E+07 1.8E+07 7.7E+05 1.2E+06 1.3E+06 TUBA4A 1.9E+05 7.7E+05 1.5E+06 2.7E+02 2.5E+02 1.7E+05 PRDX2 1.9E+06 3.7E+06 6.3E+06 1.9E+05 3.3E+04 6.5E+05 TSPAN8 2.1E+05 9.7E+04 8.3E+05 3.1E+04 6.5E+02 5.2E+04 SERPINC1 5.4E+06 2.8E+07 3.2E+07 4.2E+05 1.8E+06 2.7E+06 CLIC1 2.2E+06 2.0E+06 3.4E+06 4.6E+04 1.1E+05 4.0E+05 PGD 2.3E+05 2.5E+05 4.5E+05 5.0E+01 7.1E+04 5.0E+01 RDX 7.6E+05 2.9E+05 9.7E+05 5.0E+01 5.0E+01 1.5E+05 C6 7.7E+05 2.0E+06 2.3E+06 1.0E+05 1.1E+05 1.9E+05 TXN 6.7E+06 6.2E+06 2.0E+07 7.1E+05 4.1E+05 1.5E+06 MANF 1.9E+05 5.0E+01 2.2E+05 5.0E+01 2.6E+02 3.1E+04 HABP2 4.1E+05 5.0E+01 5.0E+01 1.7E+02 5.0E+01 3.3E+04 FBLN1 4.8E+05 7.8E+05 1.1E+05 5.0E+01 5.0E+01 1.1E+05 NT5E 8.9E+04 1.8E+06 1.2E+06 8.5E+04 1.8E+05 5.0E+01 QSOX1 1.7E+06 6.4E+05 3.3E+06 7.1E+04 1.8E+05 2.3E+05 C7 1.7E+06 4.2E+06 2.5E+06 1.4E+05 3.0E+05 2.9E+05 PON1 1.7E+06 4.6E+06 4.3E+06 1.2E+05 3.1E+05 5.1E+05 IPI00386136 1.2E+06 5.0E+01 6.1E+03 2.9E+02 5.0E+01 1.1E+05 C8A 1.3E+06 2.0E+06 1.7E+05 1.8E+02 6.0E+04 2.6E+05 KRT13 1.4E+06 3.4E+05 2.1E+06 1.0E+05 1.4E+05 1.2E+05 SLC25A5 3.7E+06 6.5E+06 6.6E+06 3.4E+04 4.8E+05 1.1E+06 HSP90AB1 8.3E+05 8.4E+05 1.4E+06 5.0E+01 6.5E+04 2.2E+05 ALPP 1.1E+05 1.0E+05 3.0E+05 4.9E+04 5.0E+01 5.0E+01 F2 5.1E+06 1.2E+07 8.5E+06 8.6E+05 6.0E+05 1.0E+06 IPI00783024 9.0E+05 2.1E+06 1.6E+05 1.4E+02 2.3E+04 2.8E+05 EPS8L3 4.3E+05 3.8E+05 8.9E+05 5.0E+01 3.1E+04 1.3E+05
19 Diabetes Page 48 of 61
PDCD6IP 2.8E+05 2.0E+05 3.7E+05 5.0E+01 5.0E+01 8.3E+04 CST3 5.9E+06 2.1E+06 2.8E+06 7.1E+05 3.3E+05 5.8E+04 GSN 5.8E+06 1.0E+07 1.1E+07 1.1E+06 4.3E+05 1.4E+06 HNRNPA2B1 8.1E+05 4.3E+05 2.3E+06 9.3E+04 1.9E+05 9.3E+04 FGA 1.4E+07 2.1E+07 2.5E+07 1.5E+05 3.0E+06 3.3E+06 HSPA1A;HSPA1B 6.4E+04 5.0E+01 2.0E+06 5.0E+01 5.1E+04 1.7E+05 HSD17B2 2.2E+06 2.3E+06 1.5E+06 5.0E+01 4.5E+04 6.2E+05 SDHB 4.6E+05 5.0E+01 1.0E+06 1.7E+04 9.7E+04 4.9E+04 RHOC 3.0E+05 3.2E+05 4.0E+05 5.0E+01 5.0E+01 1.2E+05 PPIAP19 1.9E+06 7.2E+05 1.9E+06 5.0E+01 1.0E+05 4.4E+05 IGHG1;IGHG2;IGHV4 4.9E+05 7.9E+04 2.8E+05 3.5E+02 5.0E+01 1.0E+05 -31;IGH@ GSTA3 5.0E+06 3.2E+06 2.9E+06 8.3E+04 3.1E+05 9.6E+05 F11R 4.7E+05 7.5E+05 1.7E+06 5.0E+01 1.1E+05 2.6E+05 IPI00019591 8.3E+06 2.4E+07 1.4E+07 4.0E+02 4.0E+05 5.8E+06 HADHA 2.7E+06 3.2E+06 4.0E+06 4.8E+05 2.0E+05 6.5E+05 MDH1 4.7E+05 5.0E+05 1.1E+05 2.4E+02 5.0E+01 1.5E+05 MUC13 4.1E+06 1.9E+06 5.4E+06 1.5E+05 3.4E+05 1.1E+06 CD36 1.6E+06 9.7E+05 2.5E+06 1.1E+04 3.6E+05 3.4E+05 KRT20 1.6E+06 1.7E+06 6.0E+06 2.1E+02 7.1E+04 1.2E+06 IPI00924820 5.9E+05 2.7E+05 6.4E+05 5.0E+01 3.6E+04 1.7E+05 IPI00164838 2.0E+05 2.5E+05 1.0E+05 5.0E+01 5.0E+01 7.6E+04 GSTA5 8.2E+05 5.0E+01 4.4E+05 5.0E+01 5.5E+02 1.8E+05 SOD1 5.9E+05 8.7E+05 3.2E+05 2.1E+05 4.7E+04 5.0E+01 ANXA2 2.2E+06 2.6E+06 4.2E+06 3.8E+04 5.4E+05 7.3E+05 CTSB 1.4E+05 4.1E+05 1.4E+05 5.0E+01 1.0E+05 5.0E+01 OTC 7.6E+05 5.6E+05 6.7E+05 9.8E+04 6.9E+04 1.3E+05 LRG1 1.5E+06 2.7E+06 1.3E+06 5.0E+01 2.3E+05 6.0E+05 CES2 7.9E+05 1.2E+06 1.2E+06 5.0E+01 2.2E+05 2.6E+05 LIPF 6.6E+07 4.0E+06 5.3E+07 1.4E+07 3.8E+06 1.0E+06 ACTA2 1.2E+07 1.6E+07 1.8E+07 6.5E+05 2.3E+06 4.1E+06 OLFM4 1.1E+07 1.1E+06 3.4E+06 8.4E+04 1.2E+06 1.2E+06 ACE2 1.4E+06 1.2E+06 4.2E+06 1.1E+05 9.0E+05 4.8E+04 APOE 2.4E+05 6.4E+06 2.2E+06 9.0E+05 3.3E+05 1.8E+05 PPIB 9.4E+05 7.5E+05 1.2E+06 3.4E+04 1.1E+02 4.5E+05 ECHS1 5.2E+05 1.0E+06 1.9E+06 5.0E+01 1.9E+05 3.8E+05 HPGD 2.0E+05 3.3E+05 5.8E+05 9.7E+04 3.3E+04 5.3E+04 MSMB 1.3E+05 1.0E+05 2.2E+05 7.5E+04 5.0E+01 5.0E+01 HMGCS2 2.8E+05 7.4E+05 4.0E+05 5.7E+04 5.2E+04 1.3E+05 LYZ 4.4E+07 1.5E+07 2.6E+07 6.3E+06 2.7E+06 5.7E+06 MAOA 3.2E+05 2.6E+05 6.8E+05 5.0E+01 2.1E+02 2.2E+05 GPA33 6.3E+05 4.0E+05 1.6E+06 8.5E+04 2.3E+05 1.5E+05 SUCLG1 1.1E+06 1.2E+06 1.3E+06 5.0E+01 4.8E+05 1.6E+05 PIGR 1.0E+08 4.3E+07 7.3E+07 1.4E+07 1.3E+07 1.2E+07 ATP1B3 3.7E+05 2.4E+05 3.3E+05 5.0E+01 7.4E+04 9.4E+04
20 Page 49 of 61 Diabetes
FABP5L9;FABP5L2; 5.0E+01 7.4E+01 1.3E+05 2.4E+04 5.0E+01 5.0E+01 FABP5;FABP5L7 CRISP3 6.1E+05 1.0E+05 1.9E+05 3.5E+04 1.2E+02 1.3E+05 GOLM1 1.5E+06 3.5E+05 3.6E+06 4.7E+05 1.6E+05 3.7E+05 FAM3C 5.8E+05 1.7E+05 1.1E+06 3.9E+04 1.3E+05 1.7E+05 ALDH2 3.9E+06 2.1E+06 3.0E+06 1.6E+05 7.4E+05 7.7E+05 ACAA2 4.8E+05 2.3E+05 6.9E+05 1.3E+05 9.0E+04 4.1E+04 FCN3 6.5E+05 2.0E+06 1.5E+06 5.0E+01 2.6E+05 5.3E+05 PLG 1.5E+07 2.2E+07 1.3E+07 5.5E+05 3.0E+06 5.7E+06 CLU 3.6E+06 2.0E+07 1.8E+07 4.8E+05 5.0E+06 2.5E+06 IGHM 5.6E+07 1.8E+07 2.4E+07 1.1E+06 7.1E+06 1.1E+07 FABP1 1.2E+07 1.1E+07 1.6E+07 8.1E+05 1.9E+06 5.0E+06 GC 2.1E+07 2.7E+07 2.1E+07 2.3E+05 3.6E+06 9.9E+06 GPLD1 6.4E+05 1.2E+06 1.2E+06 5.0E+01 5.0E+01 6.2E+05 CLTC 1.5E+05 1.1E+06 1.1E+06 1.5E+05 1.8E+05 1.5E+05 TUBB 8.0E+04 5.0E+01 9.7E+04 5.0E+01 1.2E+02 3.6E+04 APOB 2.1E+07 6.4E+07 8.2E+07 4.0E+06 6.1E+06 2.4E+07 PRDX1 7.3E+06 7.0E+06 1.2E+07 7.0E+05 3.1E+06 1.6E+06 YWHAE 1.1E+06 5.0E+01 3.2E+05 5.0E+01 4.3E+02 2.8E+05 MASP2 4.1E+05 1.4E+05 1.0E+05 5.0E+01 5.0E+01 1.4E+05 HEPACAM 5.0E+01 3.9E+05 5.7E+04 5.9E+04 3.5E+04 5.0E+01 KLKB1 1.9E+06 4.5E+06 1.1E+06 6.0E+05 3.4E+05 6.7E+05 IGHA1 1.8E+06 1.3E+06 2.5E+02 1.1E+02 2.2E+05 4.5E+05 SERPINA7 7.7E+05 6.1E+05 6.5E+05 5.0E+01 7.8E+04 3.7E+05 ALB 3.0E+06 3.1E+06 1.2E+06 5.0E+01 4.0E+04 1.6E+06 CFHR2 1.7E+06 4.8E+06 1.7E+06 5.0E+01 4.1E+05 1.4E+06 ANPEP 2.6E+07 1.1E+07 1.3E+07 4.3E+06 5.5E+06 1.7E+06 GIF 5.6E+05 5.0E+01 1.3E+05 1.3E+05 3.0E+04 5.0E+01 UBC;RPS27A;UBB 2.2E+06 1.6E+06 5.0E+06 1.0E+06 4.6E+05 4.9E+05 ATRN 1.2E+06 1.3E+06 7.5E+05 2.5E+04 2.1E+05 5.2E+05 C8G 1.2E+06 1.8E+06 1.1E+06 2.3E+04 1.6E+05 7.6E+05 IPI00827815 2.2E+06 1.2E+06 3.6E+06 5.0E+01 4.1E+05 1.2E+06 SPINK1 4.3E+05 6.1E+05 7.2E+05 5.0E+01 2.0E+05 2.0E+05 SI 9.8E+06 4.5E+06 1.2E+07 7.3E+05 3.2E+06 2.1E+06 IPI00386137 5.0E+01 3.8E+05 4.4E+05 5.0E+01 8.5E+04 1.0E+05 CDH17 3.7E+06 3.4E+06 1.1E+07 9.8E+05 2.0E+06 1.3E+06 MVP 4.4E+05 7.2E+05 2.6E+06 2.6E+05 4.8E+05 2.0E+05 HSP90AA2 1.6E+05 5.0E+01 1.4E+05 2.1E+02 1.6E+02 7.2E+04 APOD 1.9E+06 2.5E+06 1.9E+06 1.1E+05 3.8E+05 1.0E+06 MUC6 1.1E+08 1.6E+07 6.2E+07 6.6E+06 2.0E+07 2.1E+07 HLA-H 1.3E+05 7.5E+04 5.0E+01 1.5E+02 5.0E+01 5.2E+04 S100A9 2.4E+05 6.3E+05 7.6E+05 1.5E+05 7.1E+04 1.9E+05 LOC100126583;IGH 9.0E+05 4.2E+05 4.8E+05 5.0E+01 9.1E+04 3.7E+05 A2 SERPIND1 2.7E+06 7.0E+06 6.7E+06 6.0E+02 1.1E+06 3.2E+06
21 Diabetes Page 50 of 61
PTGR1 5.5E+05 8.4E+05 7.0E+05 3.3E+05 1.4E+05 7.9E+04 GDI2 7.8E+05 1.7E+06 3.6E+06 2.7E+05 9.6E+05 3.7E+05 TUBA1C 1.5E+06 1.3E+06 3.4E+06 5.2E+04 5.3E+05 1.0E+06 CD5L 8.6E+06 6.0E+06 5.5E+06 7.7E+04 7.5E+05 4.5E+06 SERPINA1 1.1E+07 2.9E+07 2.9E+07 2.1E+05 6.1E+06 1.2E+07 DAK 1.3E+06 2.2E+06 1.3E+06 1.1E+06 4.7E+04 1.2E+05 SERPINA4 6.3E+05 3.4E+06 5.3E+06 9.1E+04 1.1E+06 1.3E+06 PPP1CC 2.0E+05 2.2E+05 2.6E+05 6.3E+04 7.0E+04 5.1E+04 REG3A 2.9E+06 4.5E+06 6.8E+06 9.1E+05 6.9E+05 2.2E+06 F13B 5.5E+06 3.0E+06 3.0E+06 5.0E+01 4.1E+05 2.7E+06 ACTN4 6.6E+06 4.3E+06 8.0E+06 6.3E+05 9.8E+05 3.6E+06 PDIA6 1.2E+05 5.0E+01 7.6E+04 1.7E+02 8.9E+01 5.4E+04 IPI00382439 8.1E+05 9.4E+05 1.4E+06 5.0E+01 1.5E+05 7.3E+05 IPI00384401 4.3E+06 4.0E+06 4.7E+06 4.1E+04 2.3E+05 3.4E+06 ANXA2P2 1.9E+06 1.6E+06 4.1E+06 9.8E+05 5.7E+05 6.3E+05 PARK7 5.8E+05 5.1E+05 7.1E+05 1.8E+05 5.0E+04 2.9E+05 DPP4 2.3E+05 6.7E+05 6.4E+05 3.6E+04 1.6E+05 2.5E+05 CANX 2.2E+06 1.4E+06 7.2E+06 1.4E+06 1.1E+06 6.1E+05 ACTBL2 5.8E+06 6.2E+06 4.8E+06 4.4E+05 1.8E+06 2.7E+06 ACY1 8.7E+05 2.4E+06 1.2E+06 1.3E+05 9.3E+05 2.3E+05 IPI00385264 8.6E+07 1.1E+07 1.4E+07 3.5E+06 1.2E+07 1.7E+07 IPI00830057 1.6E+05 2.3E+05 4.6E+05 5.0E+01 4.8E+04 2.0E+05 SERPING1 3.8E+06 7.6E+06 7.1E+06 1.0E+05 1.8E+06 3.5E+06 S100A8 1.7E+06 1.1E+06 5.0E+06 1.6E+06 3.5E+05 2.9E+05 ETFDH 1.8E+05 5.0E+01 1.9E+05 5.6E+04 5.2E+04 5.0E+01 P4HB 1.6E+07 9.0E+06 1.2E+07 1.6E+06 5.5E+06 3.7E+06 LOC440786 5.1E+05 5.0E+01 2.5E+05 5.0E+02 3.1E+02 2.2E+05 KRT24 1.9E+05 5.0E+01 4.5E+02 1.7E+02 5.0E+01 5.5E+04 CSTB 1.2E+06 2.8E+06 1.4E+06 1.2E+06 2.9E+05 1.6E+05 COX4I1 5.4E+05 8.0E+05 1.2E+05 3.1E+05 1.1E+05 1.8E+04 IPI00827690 3.1E+05 1.3E+05 3.4E+05 5.0E+01 3.7E+04 2.0E+05 C1R 1.7E+06 5.8E+06 4.4E+06 8.1E+04 9.9E+05 2.6E+06 IGKC 5.8E+05 5.0E+01 2.3E+05 5.0E+01 5.3E+04 2.0E+05 C1S 2.0E+06 6.3E+06 3.1E+06 5.0E+05 1.1E+06 1.9E+06 IPI00854709 3.4E+05 2.0E+05 2.7E+05 5.0E+01 5.0E+01 2.5E+05 RAB1A 1.2E+06 1.3E+06 2.3E+06 6.5E+05 3.8E+05 4.4E+05 EEF1A2 2.2E+06 2.7E+06 5.4E+06 8.7E+05 7.0E+05 1.7E+06 IGKV3D-11 1.7E+06 1.7E+06 1.4E+06 1.5E+04 3.9E+05 1.1E+06 LOC100126583;IGH 4.3E+07 1.0E+07 7.6E+06 4.1E+06 8.4E+06 6.8E+06 A2 A1BG 8.0E+06 1.6E+07 1.2E+07 1.2E+05 3.7E+06 7.7E+06 PDIA4 5.5E+05 6.3E+05 7.6E+05 1.8E+05 3.1E+05 1.4E+05 IGKV3-20 1.6E+07 1.9E+07 3.1E+06 4.5E+05 2.8E+06 9.2E+06 IGLV7-43 5.0E+05 2.3E+05 1.6E+05 5.0E+01 1.1E+05 1.8E+05 LOC100126583;IGH 1.7E+06 1.6E+06 5.0E+01 5.0E+01 3.5E+05 7.5E+05
22 Page 51 of 61 Diabetes
A2 PGK1 2.7E+06 1.7E+06 3.2E+06 8.7E+04 7.5E+05 1.7E+06 ACTB 6.8E+06 8.0E+06 1.2E+07 1.7E+05 1.7E+06 7.1E+06 IPI00382494 5.6E+06 3.0E+06 2.2E+06 5.0E+01 9.7E+05 2.7E+06 ORM2 7.2E+06 2.1E+07 8.1E+06 1.8E+05 3.3E+06 8.9E+06 PSMB8 5.5E+05 3.9E+05 2.3E+05 5.0E+01 2.8E+05 1.4E+05 IPI00735451 3.8E+06 3.5E+06 4.7E+06 7.7E+05 8.2E+05 2.7E+06 HADHB 4.1E+06 2.9E+06 3.5E+06 9.9E+05 1.2E+06 1.6E+06 PTGDS 6.1E+05 2.2E+06 7.0E+05 5.0E+01 7.1E+05 5.5E+05 IGHA1 1.1E+07 5.6E+06 5.5E+06 1.1E+06 3.4E+06 3.5E+06 MME 1.7E+06 6.8E+05 1.6E+06 3.1E+05 6.0E+05 5.3E+05 ATP1A1 1.5E+06 9.3E+05 1.5E+06 9.6E+05 1.4E+05 3.1E+05 ACTN1 7.8E+05 1.8E+06 7.7E+05 3.4E+05 3.2E+05 5.8E+05 HSPE1 3.3E+06 1.3E+06 1.9E+06 7.9E+05 9.6E+05 6.8E+05 CAP1 1.5E+06 1.2E+06 1.9E+06 5.0E+01 9.4E+05 7.6E+05 PLS1 6.6E+06 2.9E+06 5.5E+06 2.8E+06 1.2E+06 1.6E+06 IGHG1;IGHG2;IGHV4 3.0E+06 2.4E+06 3.1E+06 6.8E+02 5.0E+05 2.7E+06 -31;IGH@ IPI00783094 3.3E+05 2.0E+05 6.4E+05 5.0E+01 1.2E+05 3.2E+05 HP 3.9E+07 1.1E+08 5.5E+07 2.6E+06 1.5E+07 5.9E+07 AKR1C4 1.9E+06 1.5E+06 1.6E+06 8.3E+05 1.3E+05 9.1E+05 IPI00382500 1.6E+06 1.8E+06 2.3E+06 3.4E+05 8.0E+05 1.0E+06 PROS1 3.9E+06 5.2E+06 3.9E+06 7.1E+04 1.5E+06 3.4E+06 SPRR1A 7.4E+05 3.4E+05 3.3E+06 1.6E+06 5.8E+04 5.0E+01 IPI00387100 2.5E+05 3.9E+05 3.6E+05 6.6E+04 1.3E+05 1.8E+05 DMBT1 4.3E+07 5.2E+06 6.7E+06 1.6E+07 3.2E+06 1.9E+06 IPI00069693 3.6E+05 5.0E+01 4.7E+05 5.0E+01 6.1E+02 3.2E+05 VIL1 5.8E+06 3.6E+06 7.3E+06 4.1E+05 1.5E+06 4.5E+06 ORM1 3.2E+06 2.4E+07 4.1E+06 5.2E+06 2.5E+06 4.2E+06 CD14 4.5E+04 2.7E+06 6.3E+05 2.4E+05 4.6E+05 6.1E+05 IPI00827944 4.5E+05 2.4E+05 1.6E+05 5.0E+01 8.4E+04 2.5E+05 PREP 3.3E+05 1.5E+05 4.2E+05 9.5E+04 1.1E+05 1.4E+05 SERPINA3 1.8E+07 1.3E+07 1.0E+07 4.4E+05 4.0E+06 1.2E+07 LOC100126583;IGH 2.3E+06 8.9E+05 1.3E+05 1.7E+02 5.0E+05 8.3E+05 A2 RBP2 1.6E+06 1.6E+06 1.7E+06 2.6E+05 1.0E+06 7.0E+05 IPI00387026 2.0E+06 6.6E+05 4.4E+05 2.1E+04 4.1E+05 8.1E+05 PPIA 5.0E+01 1.1E+02 1.1E+05 4.4E+04 1.3E+02 5.0E+01 SPRR2C;SPRR2G 4.8E+05 1.2E+02 2.0E+05 2.2E+05 4.6E+04 5.0E+01 PCK2 5.8E+06 3.4E+06 2.4E+06 1.7E+06 1.6E+06 1.3E+06 IPI00382436 4.3E+05 7.3E+05 6.8E+05 5.0E+01 8.1E+04 6.7E+05 CP 2.5E+07 4.4E+07 3.3E+07 3.3E+06 8.8E+06 3.0E+07 RAP1B 1.4E+05 7.1E+04 2.4E+05 4.8E+04 7.5E+04 5.8E+04 CES1 1.4E+05 5.0E+01 2.2E+05 5.0E+01 2.9E+02 1.5E+05 HSPA8 5.6E+06 2.2E+06 6.2E+06 1.8E+06 1.1E+06 2.9E+06
23 Diabetes Page 52 of 61
CFHR5 2.3E+05 2.5E+05 4.1E+05 5.0E+01 4.1E+02 3.7E+05 KRT3 6.7E+05 3.0E+05 1.5E+06 7.7E+05 2.9E+02 2.6E+05 LDHAL6A 3.2E+06 3.6E+06 6.8E+05 6.8E+03 1.6E+06 1.5E+06 IPI00384409 3.2E+05 5.0E+05 1.4E+05 5.0E+01 7.2E+04 3.2E+05 IGLL1 4.5E+06 1.3E+06 7.5E+05 3.3E+05 1.0E+06 1.4E+06 ITIH4 1.1E+07 2.0E+07 2.0E+07 1.3E+06 5.9E+06 1.4E+07 PYCARD 5.0E+01 5.0E+01 2.6E+02 5.0E+01 5.0E+01 5.0E+01 LOC284422 2.8E+05 1.3E+05 1.4E+05 5.0E+01 5.0E+01 2.3E+05 TFF1 2.0E+06 4.1E+05 1.8E+06 1.5E+06 2.4E+05 2.0E+04 CAPRIN2 1.6E+05 1.4E+05 6.0E+04 5.0E+01 4.5E+04 1.0E+05 IPI00382420 1.2E+06 8.4E+05 8.9E+04 3.8E+02 5.0E+01 9.1E+05 CPB2 2.2E+05 2.2E+05 2.7E+05 5.0E+01 1.3E+05 1.7E+05 GMDS 1.9E+05 2.9E+05 6.5E+05 1.2E+05 2.9E+05 7.3E+04 ITLN2 1.2E+06 2.0E+06 3.5E+06 1.2E+06 1.2E+06 4.9E+05 CBR1 3.8E+06 5.3E+06 9.0E+06 5.2E+06 1.5E+06 1.1E+06 ATP1B1 1.5E+06 5.0E+05 1.9E+06 4.0E+05 4.5E+05 8.6E+05 VTN 2.1E+06 1.1E+07 9.3E+06 7.6E+05 3.7E+06 5.6E+06 TP53I3 1.7E+05 5.0E+01 5.0E+01 2.3E+02 5.0E+01 7.4E+04 PRKCSH 8.9E+04 5.0E+01 1.9E+05 5.0E+01 7.4E+04 5.2E+04 EPCAM 1.6E+06 5.7E+05 1.7E+06 2.8E+04 6.4E+05 1.0E+06 VDAC1 2.8E+06 1.2E+06 8.6E+06 1.3E+06 2.5E+06 1.8E+06 CPT2 6.1E+05 1.8E+05 5.8E+05 5.0E+01 4.8E+05 1.3E+05 IGKV1-5 3.5E+05 1.6E+05 1.5E+05 5.0E+01 7.4E+04 2.2E+05 AKR1C3 9.0E+04 7.2E+05 1.5E+06 9.8E+05 5.0E+01 5.3E+04 TUBB2C 9.8E+05 4.2E+05 1.8E+06 4.0E+04 5.0E+05 9.0E+05 C5 5.1E+06 9.6E+06 8.2E+06 5.1E+05 3.6E+06 6.3E+06 FN1 1.6E+07 2.2E+07 1.4E+07 4.3E+05 4.6E+06 1.8E+07 ALDH1A1 7.1E+06 8.7E+06 1.6E+07 6.8E+06 5.5E+06 2.6E+06 ANP32B 8.8E+04 9.6E+04 3.6E+02 5.0E+01 8.7E+04 5.0E+01 GDA 6.6E+05 8.1E+05 2.5E+06 7.3E+05 5.0E+05 6.7E+05 IPI00829653 1.2E+05 6.7E+04 5.0E+01 2.5E+02 5.0E+01 9.0E+04 UGT2B17 2.5E+06 6.9E+05 3.4E+06 2.6E+06 1.5E+02 5.9E+05 IPI00829701 9.2E+05 2.5E+05 3.2E+05 1.8E+02 1.3E+05 5.8E+05 IGHA1 1.3E+08 5.4E+07 7.0E+07 2.3E+07 5.1E+07 4.7E+07 VCP 7.6E+05 4.2E+05 1.8E+06 1.4E+05 6.3E+05 6.9E+05 CEACAM5 1.4E+05 1.1E+05 1.1E+05 5.2E+04 3.4E+04 8.9E+04 GSTA1 3.2E+06 1.2E+06 2.7E+06 2.7E+06 1.2E+03 8.5E+05 EEF1G 3.1E+05 1.0E+06 2.3E+06 5.9E+05 9.9E+05 2.8E+05 LCP1 6.4E+05 2.0E+05 3.8E+05 1.3E+05 6.4E+04 4.3E+05 IGLC1;IGLV1- 8.6E+06 5.4E+06 5.9E+06 1.0E+05 2.9E+06 7.2E+06 44;IGLV1-40;IGLV3- 21;IGLV2-11;IGLV2- 14;IGL@;IGLC2;IGLC 3 FGG 7.2E+07 4.2E+07 1.8E+07 3.3E+06 1.9E+07 4.5E+07
24 Page 53 of 61 Diabetes
ALDOA 1.7E+05 6.9E+05 4.5E+05 5.0E+01 6.2E+05 5.6E+04 LGALS3BP 6.1E+06 1.4E+06 1.1E+06 8.2E+05 2.3E+06 1.3E+06 IPI00385253 7.1E+05 3.2E+05 6.4E+05 1.2E+02 2.0E+05 6.7E+05 SDHA 9.7E+05 4.4E+05 9.3E+05 1.6E+05 9.8E+05 6.8E+04 MDH2 2.5E+06 1.1E+06 1.4E+06 1.1E+06 1.2E+06 3.5E+05 IPI00829873 2.1E+05 5.0E+01 1.3E+05 5.0E+01 1.8E+02 1.8E+05 ADH5;ADH5P4 5.0E+01 1.9E+05 3.6E+05 4.5E+04 2.4E+05 5.0E+01 CA2 1.2E+06 3.4E+06 7.0E+06 2.9E+06 2.5E+06 8.8E+05 IPI00101961 7.3E+05 1.2E+05 2.4E+05 1.8E+05 2.2E+05 1.9E+05 IGLV2-18 5.9E+05 1.4E+05 7.5E+04 4.5E+04 3.3E+05 6.0E+04 APOH 4.9E+06 2.8E+07 7.3E+06 1.3E+06 8.4E+06 1.2E+07 LOC390956 5.6E+05 1.1E+02 1.6E+06 3.6E+05 2.5E+05 5.7E+05 IPI00003939 5.2E+05 5.6E+05 1.9E+05 5.0E+01 8.0E+04 6.2E+05 MYL12B 5.0E+01 5.0E+05 1.2E+06 7.6E+05 1.7E+05 5.0E+01 CFH 1.6E+07 2.3E+07 2.4E+07 1.2E+06 8.8E+06 2.4E+07 DECR1 4.0E+05 5.0E+01 2.9E+05 7.1E+04 2.2E+05 8.2E+04 LOC401847 1.8E+06 8.2E+05 4.0E+05 5.0E+01 3.4E+05 1.4E+06 PRSS7 5.9E+06 2.8E+06 8.2E+06 5.0E+06 1.6E+06 2.6E+06 NUDT21 2.6E+05 1.0E+05 2.8E+05 2.1E+05 1.4E+05 1.9E+04 LOC652128 1.0E+07 4.3E+06 3.2E+06 3.5E+02 3.6E+06 6.6E+06 IGLC1;IGLV1- 3.6E+07 1.7E+07 2.0E+07 1.1E+06 1.4E+07 2.7E+07 44;IGLV1-40;IGLV3- 21;IGLV2-11;IGLV2- 14;IGL@;IGLC2;IGLC 3 A2M 9.7E+07 1.0E+08 1.1E+08 8.6E+06 6.1E+07 1.1E+08 TFF3 2.0E+06 1.4E+06 4.8E+06 2.0E+06 1.9E+06 8.6E+05 ENO1 4.9E+06 3.9E+06 4.9E+06 1.5E+06 2.8E+06 3.8E+06 TCN1 1.5E+05 3.0E+05 6.8E+05 4.0E+04 1.1E+05 5.1E+05 IGLC1;IGLV1- 5.0E+01 5.0E+01 9.6E+05 2.0E+02 1.2E+03 5.8E+05 44;IGLV1-40;IGLV3- 21;IGLV2-11;IGLV2- 14;IGL@;IGLC2;IGLC 3 C20orf114 2.0E+06 1.5E+05 3.8E+05 7.4E+05 1.9E+05 6.0E+05 IPI00382478 5.0E+06 2.6E+06 1.4E+06 1.4E+02 1.7E+06 3.8E+06 IGHG1;IGHG2;IGHV4 5.0E+01 5.5E+05 1.6E+06 5.0E+01 1.3E+06 5.0E+01 -31;IGH@ IPI00760721 9.6E+05 6.4E+05 1.0E+05 3.0E+04 4.4E+05 5.8E+05 MUC2 2.2E+07 4.1E+06 1.2E+07 4.3E+06 1.1E+07 8.6E+06 FH 8.4E+05 4.8E+05 1.2E+06 7.9E+05 6.3E+05 1.3E+05 AZGP1 7.0E+06 1.2E+07 4.9E+06 4.1E+06 4.3E+06 6.6E+06 HDGF 3.7E+06 1.9E+05 1.9E+05 5.0E+01 5.0E+01 2.6E+06 SELENBP1 6.2E+04 6.5E+05 4.8E+05 5.0E+01 1.0E+05 6.5E+05 IPI00383732 2.8E+06 2.5E+06 1.3E+06 6.2E+02 1.3E+06 3.0E+06 IGHA1 1.0E+06 8.0E+05 1.1E+06 5.0E+01 2.4E+05 1.6E+06
25 Diabetes Page 54 of 61
BCHE 5.1E+05 6.2E+05 1.0E+06 3.2E+04 5.3E+05 7.9E+05 DLD 6.5E+05 1.7E+05 5.3E+05 2.3E+05 5.4E+05 8.3E+04 IGLV8-61 7.5E+05 1.6E+05 2.0E+05 3.7E+02 1.9E+05 5.2E+05 WDR1 2.0E+06 1.4E+06 1.5E+06 9.4E+05 1.1E+06 1.1E+06 GALNT7 2.9E+05 1.4E+05 5.2E+05 4.2E+05 8.3E+04 1.1E+05 LAP3 9.7E+05 8.3E+05 6.2E+05 1.1E+05 7.6E+05 6.9E+05 HEBP1 2.0E+06 1.2E+06 1.4E+06 2.2E+05 2.1E+06 6.8E+05 HSPA5 6.1E+06 5.9E+06 8.2E+06 6.4E+06 4.1E+06 2.7E+06 NME1;NME2 1.0E+06 5.0E+01 6.3E+05 1.1E+05 4.4E+05 5.4E+05 ADH6 1.4E+05 5.9E+05 1.3E+06 1.7E+05 9.7E+05 2.2E+05 C4BPA 5.2E+06 6.9E+06 4.7E+06 4.0E+05 3.3E+06 7.5E+06 IPI00550731 5.3E+07 5.7E+07 6.6E+07 2.7E+07 3.4E+07 5.6E+07 HBZ 2.0E+06 1.3E+06 1.9E+06 5.0E+01 6.3E+05 2.9E+06 IPI00830122 1.2E+05 9.6E+04 5.0E+01 5.0E+01 4.0E+04 1.0E+05 CNDP2 9.8E+05 1.4E+06 1.8E+06 1.8E+06 5.4E+05 5.5E+05 ACADVL 1.2E+06 8.4E+05 2.3E+05 2.3E+05 1.2E+06 1.2E+05 IGKV4-1 4.0E+06 4.3E+06 6.1E+06 1.5E+06 2.4E+06 5.9E+06 TF 8.8E+07 1.4E+08 8.4E+07 1.2E+07 8.1E+07 1.2E+08 IGHG4 1.0E+07 2.5E+07 1.6E+07 8.1E+05 5.7E+06 2.8E+07 HPR 7.9E+06 1.4E+07 9.0E+06 8.8E+05 5.3E+06 1.5E+07 IPI00382486 3.3E+05 8.6E+04 6.2E+04 5.0E+01 7.7E+04 2.5E+05 ACE 8.3E+05 1.4E+05 7.1E+05 1.5E+05 3.4E+05 6.9E+05 IPI00007899 4.3E+06 8.7E+05 4.5E+05 1.5E+05 1.2E+06 2.5E+06 MYL6B 1.9E+06 6.8E+05 1.2E+06 1.6E+05 1.5E+06 1.0E+06 SERPINA1 4.1E+07 8.3E+07 5.0E+07 1.6E+07 3.6E+07 7.3E+07 AKR1A1 3.6E+05 7.9E+05 8.1E+05 6.1E+05 7.5E+05 5.8E+04 TTR 1.5E+07 1.6E+07 5.1E+06 2.0E+06 6.3E+06 1.8E+07 ALB 5.9E+08 7.8E+08 5.3E+08 1.0E+08 4.0E+08 8.9E+08 HPR;HP 1.5E+07 2.2E+07 1.1E+07 1.3E+06 9.8E+06 2.4E+07 CALR 4.1E+06 2.7E+06 4.5E+06 4.7E+06 2.7E+06 9.8E+05 PGM2 4.3E+05 2.5E+05 4.4E+05 3.5E+05 3.1E+05 1.7E+05 UGT2B15 2.3E+05 2.7E+05 1.5E+06 1.4E+06 3.0E+02 1.1E+05 IPI00029863 1.9E+06 4.0E+06 1.7E+06 1.7E+06 2.5E+06 1.5E+06 HBB 9.4E+06 1.8E+07 3.1E+07 4.0E+05 2.0E+07 2.4E+07 IPI00003470 1.8E+06 1.9E+05 2.1E+05 7.5E+02 1.6E+05 1.5E+06 ADA 1.1E+07 7.1E+06 5.5E+06 3.8E+06 7.6E+06 6.7E+06 CLCA1 3.9E+07 1.5E+07 4.1E+07 3.8E+06 4.5E+07 2.5E+07 SLPI 1.1E+07 1.8E+05 8.1E+06 1.4E+07 2.9E+05 7.2E+05 IGHG1;IGHG2;IGHV4 3.6E+05 3.5E+05 2.0E+05 1.4E+05 2.0E+05 3.6E+05 -31;IGH@ SERPINA6 1.3E+06 2.4E+06 6.6E+05 5.0E+01 1.2E+06 2.2E+06 CS 3.0E+05 3.1E+02 5.0E+01 1.2E+05 5.0E+01 1.1E+05 GOT2 2.3E+05 1.9E+05 3.8E+05 1.9E+05 1.9E+05 2.5E+05 HSPB1 2.3E+05 5.0E+01 6.5E+05 1.2E+05 4.8E+05 8.2E+04 KRT6B 7.7E+05 3.2E+03 2.2E+06 1.2E+06 4.0E+05 7.2E+05
26 Page 55 of 61 Diabetes
IPI00384397 7.1E+06 1.5E+06 1.4E+06 7.3E+04 2.3E+06 5.6E+06 LOC651751 5.0E+01 3.4E+05 3.6E+05 3.6E+05 2.0E+05 5.0E+01 HPX 2.7E+07 4.1E+07 1.2E+07 1.6E+06 2.0E+07 4.2E+07 IPI00386131 2.7E+05 8.5E+04 5.0E+01 8.2E+02 5.0E+01 2.9E+05 IGHG1;IGHG2;IGHV4 5.1E+07 3.1E+07 3.8E+07 4.2E+06 2.3E+07 7.2E+07 -31;IGH@ PEBP1 6.2E+06 6.3E+06 6.9E+06 1.8E+06 2.4E+06 1.2E+07 PCK1 3.6E+05 6.2E+05 1.0E+06 4.5E+05 8.8E+05 3.5E+05 IPI00383887 1.7E+05 9.3E+04 5.0E+01 5.0E+01 5.8E+04 1.6E+05 GSR 8.2E+05 5.3E+05 8.5E+05 5.5E+05 7.5E+05 5.3E+05 ITLN1 3.3E+06 2.5E+06 1.2E+06 4.4E+05 3.0E+06 2.4E+06 C3 6.1E+07 8.0E+07 8.8E+07 9.4E+06 6.8E+07 1.2E+08 LOC653879 2.1E+07 2.7E+07 2.3E+07 5.3E+06 1.9E+07 3.8E+07 IPI00890703 3.0E+05 1.2E+05 2.8E+05 2.1E+02 5.9E+04 5.4E+05 MTTP 6.5E+06 2.1E+06 3.6E+06 3.1E+06 5.9E+06 1.6E+06 AMBP 1.0E+06 2.9E+06 2.8E+06 7.7E+04 3.1E+06 2.7E+06 IPI00382499 1.1E+06 8.5E+05 2.4E+06 1.6E+06 6.3E+05 1.5E+06 LTF 2.3E+06 3.0E+06 3.3E+06 3.8E+06 3.3E+06 5.9E+05 CFHR3 7.3E+05 5.3E+05 1.2E+05 1.8E+05 2.6E+05 8.0E+05 PEPD 2.3E+06 2.7E+05 6.3E+05 1.8E+06 5.0E+01 1.1E+06 UBA1 5.0E+01 5.0E+01 7.0E+04 5.0E+01 9.2E+01 6.3E+04 IPI00829980 7.3E+05 2.7E+05 4.5E+05 5.0E+01 2.3E+05 1.1E+06 KRT14 4.5E+05 2.4E+05 7.8E+05 7.2E+05 4.1E+05 2.3E+05 ITGB1 9.0E+05 2.7E+05 1.7E+05 4.7E+05 5.0E+05 2.7E+05 IPI00007906 6.2E+05 5.8E+05 2.2E+05 1.1E+05 3.9E+05 8.1E+05 GOT1 2.6E+05 2.8E+05 8.5E+04 6.8E+04 3.1E+05 2.0E+05 SERPINA5 2.0E+06 1.3E+06 1.1E+06 2.0E+05 1.3E+06 2.6E+06 LDHA 6.5E+05 3.5E+05 5.4E+05 1.1E+02 7.9E+05 6.6E+05 IPI00384404 1.2E+05 8.4E+04 2.6E+05 5.0E+01 1.1E+05 3.4E+05 IPI00384391 2.3E+06 4.4E+05 5.7E+05 4.2E+04 8.1E+05 2.4E+06 GNB2 5.7E+05 5.0E+01 9.6E+04 1.5E+05 3.2E+05 1.9E+05 PZP 1.1E+07 1.3E+07 1.1E+07 5.0E+06 9.4E+06 2.1E+07 CKMT1B;LOC10013 6.5E+05 2.4E+05 4.4E+05 6.3E+05 5.6E+05 1.4E+05 3623;CKMT1A C6orf58 3.5E+06 5.9E+05 4.3E+06 2.1E+06 3.4E+06 3.0E+06 GNB1 5.0E+01 1.2E+05 2.6E+05 3.3E+05 5.5E+04 5.0E+01 IGHG1;IGHG2;IGHV4 1.4E+05 5.0E+01 5.0E+01 4.1E+02 5.0E+01 1.4E+05 -31;IGH@ LOC652113 2.1E+05 5.0E+01 1.2E+06 9.8E+05 3.9E+05 8.9E+04 ANXA5 7.5E+05 5.0E+01 1.6E+05 2.0E+05 3.0E+05 4.4E+05 HBD 1.1E+07 1.4E+07 2.9E+07 4.0E+05 1.9E+07 3.6E+07 EFHD2 1.9E+05 5.0E+01 1.5E+05 2.6E+05 1.6E+04 8.2E+04 ZG16B 2.1E+06 2.4E+06 4.5E+06 5.4E+06 1.6E+06 2.5E+06 IGKV1D-8 2.5E+06 1.7E+06 1.3E+06 3.5E+05 1.6E+06 3.8E+06 UGT2B10 6.9E+05 2.1E+05 6.6E+05 1.5E+06 5.0E+01 1.6E+05
27 Diabetes Page 56 of 61
FGB 6.2E+07 2.6E+07 1.6E+07 6.8E+06 4.9E+07 5.6E+07 ANXA4 5.4E+05 7.0E+05 1.3E+06 1.8E+06 5.6E+05 3.5E+05 GSTP1 2.6E+06 2.6E+06 6.4E+06 9.5E+06 1.7E+06 1.3E+06 ALDOC 5.2E+05 5.2E+05 9.7E+05 1.7E+06 2.8E+05 2.0E+05 FLNB 4.0E+06 1.4E+06 3.0E+06 2.7E+06 3.7E+06 2.9E+06 MASP1 3.5E+05 2.1E+05 1.1E+05 3.0E+04 1.5E+02 7.1E+05 UGCGL1 2.3E+05 1.2E+05 7.2E+04 5.0E+01 1.0E+05 3.5E+05 CNDP2 6.3E+05 7.0E+05 1.1E+06 1.2E+06 4.7E+05 1.0E+06 DEFA1;LOC728358 3.2E+06 4.2E+06 2.2E+06 1.0E+07 1.3E+05 2.0E+05 IGHG3 8.3E+07 7.6E+07 4.8E+07 1.5E+07 5.2E+07 1.6E+08 IGLV3-12 2.3E+05 5.0E+01 5.0E+01 5.0E+01 5.0E+01 2.6E+05 SCARB2 4.6E+05 2.0E+05 8.2E+05 1.1E+06 5.1E+05 1.2E+05 TPI1;RCTPI1 6.8E+05 3.3E+05 4.9E+05 1.6E+05 8.9E+05 6.6E+05 OGDH 3.5E+05 7.1E+05 1.1E+06 1.4E+06 9.4E+05 1.4E+05 C4A 1.9E+07 4.4E+07 2.8E+07 3.4E+06 4.5E+07 5.9E+07 UGT1A1;UGT1A8 4.5E+05 5.5E+05 5.0E+05 6.6E+05 3.6E+05 7.5E+05 PIP 5.0E+01 2.8E+05 8.6E+05 1.2E+06 1.4E+05 5.0E+01 VDAC2 3.0E+05 8.7E+04 6.2E+05 6.1E+05 5.9E+05 5.0E+01 CBR3 1.9E+06 1.9E+06 1.5E+06 4.5E+06 1.2E+06 6.6E+05 MUC5AC 4.1E+07 6.1E+06 5.7E+07 1.0E+08 1.5E+07 7.3E+06 C1QB 1.8E+05 1.6E+05 2.3E+04 3.6E+02 5.0E+01 4.4E+05 UGT2A3 9.1E+05 2.0E+05 9.1E+04 8.1E+05 3.7E+05 2.9E+05 IPI00743194 1.9E+06 3.7E+05 4.1E+05 3.5E+05 6.2E+05 2.4E+06 MAOB 3.1E+05 6.0E+04 3.1E+05 1.9E+05 3.3E+05 3.3E+05 RBP4 4.5E+06 5.9E+06 2.6E+06 2.5E+05 6.1E+06 1.0E+07 CA1 1.1E+06 2.7E+06 4.2E+06 4.8E+05 5.3E+06 4.4E+06 PSMA6 4.6E+05 1.6E+05 1.6E+06 2.2E+05 1.8E+06 7.8E+05 ANO10 5.0E+01 1.1E+05 1.5E+05 1.4E+05 1.9E+05 5.0E+01 HLA-DRB3;HLA- 5.0E+01 1.4E+05 8.9E+04 9.1E+04 2.1E+05 5.0E+01 DRB5;LOC649783;L OC100133484;LOC1 00133661;HLA- DRB4;LOC10013381 1;HLA-DRB1 IPI00384407 1.8E+06 4.5E+05 5.5E+05 2.3E+02 8.8E+05 2.7E+06 FKBP1A 1.3E+05 1.7E+05 3.0E+05 4.2E+04 2.2E+05 5.3E+05 ENPP7 1.9E+05 5.0E+01 5.0E+01 4.9E+04 1.7E+05 3.1E+04 ACADS 6.8E+05 3.5E+05 6.2E+05 4.9E+05 1.5E+06 1.8E+05 GKN2 5.0E+01 5.0E+01 1.8E+05 9.1E+04 1.5E+05 5.0E+01 GPI 2.0E+06 1.1E+06 1.1E+06 2.9E+06 1.7E+06 9.9E+05 DDT 6.1E+05 3.9E+05 1.1E+06 1.1E+06 7.6E+05 9.6E+05 IGHG1;IGHG2;IGHV4 5.4E+05 6.8E+04 1.1E+05 3.1E+02 1.4E+05 8.4E+05 -31;IGH@ ESD 9.7E+04 5.0E+01 1.7E+05 6.9E+04 1.5E+05 1.6E+05 TKT 1.5E+06 7.3E+05 1.8E+06 1.8E+06 2.6E+06 1.5E+06
28 Page 57 of 61 Diabetes
IGHG1;IGHG2;IGHV4 1.1E+07 1.7E+07 7.9E+06 4.2E+06 1.7E+07 3.2E+07 -31;IGH@ IGLC1;IGLV1- 1.7E+05 5.0E+01 5.0E+01 5.0E+01 3.7E+04 2.1E+05 44;IGLV1-40;IGLV3- 21;IGLV2-11;IGLV2- 14;IGL@;IGLC2;IGLC 3 TGM3 3.8E+06 2.7E+05 1.3E+06 7.8E+06 1.4E+05 1.4E+05 KRT10 4.9E+06 4.0E+06 1.2E+06 1.0E+06 8.1E+06 6.4E+06 IPI00385252 1.1E+05 5.0E+01 5.0E+01 4.8E+02 5.0E+01 1.7E+05 NDUFAB1 5.5E+04 5.0E+01 1.5E+05 1.8E+05 1.6E+05 5.0E+01 IPI00829827 1.8E+06 2.0E+05 2.2E+05 5.1E+04 8.7E+05 2.6E+06 KRT5 2.4E+06 6.7E+05 1.5E+06 4.7E+06 1.5E+06 1.2E+06 ADH4 1.3E+06 1.0E+06 2.1E+06 1.7E+06 4.7E+06 8.9E+05 IGKV2-40 1.5E+06 4.6E+05 6.7E+05 8.3E+05 9.8E+05 2.5E+06 HSD17B4 1.2E+06 4.0E+05 9.2E+05 2.3E+06 1.2E+06 6.3E+05 CAT 2.0E+06 1.2E+06 1.2E+06 1.3E+06 3.0E+06 3.1E+06 ADH1A 6.7E+06 4.7E+06 9.5E+06 4.1E+06 2.0E+07 1.0E+07 FGL1 8.8E+04 4.5E+05 1.1E+05 5.9E+04 6.3E+05 4.0E+05 CMBL 2.2E+06 1.5E+06 2.4E+06 4.3E+06 3.9E+06 2.0E+06 PSCA 1.1E+05 4.6E+03 1.4E+06 2.0E+06 4.1E+04 5.5E+05 CKMT2 2.0E+05 3.2E+05 1.1E+06 4.4E+05 2.1E+06 2.5E+05 IGHG1;IGHG2;IGHV4 2.0E+05 1.8E+05 1.6E+05 1.9E+05 2.6E+05 4.9E+05 -31;IGH@ REG1A 9.7E+06 3.5E+06 1.0E+07 1.1E+07 2.1E+07 8.7E+06 AKR1C2 1.9E+06 3.3E+06 4.5E+06 1.3E+07 2.3E+06 2.0E+06 LOC652694 3.5E+06 1.5E+06 1.2E+06 1.0E+06 3.4E+06 6.4E+06 KRT16 1.1E+06 8.4E+04 4.8E+04 1.9E+06 1.9E+05 1.4E+05 LOC100133944 6.8E+05 8.5E+05 1.9E+06 3.9E+05 4.6E+06 1.1E+06 LOC652493 9.4E+04 5.0E+01 1.2E+05 1.8E+02 6.5E+04 3.2E+05 CTSD 1.2E+06 5.7E+05 1.3E+06 3.1E+06 1.8E+06 6.4E+05 FCGBP 3.4E+07 1.6E+07 2.4E+07 3.4E+07 6.6E+07 3.4E+07 HBA1;HBA2 2.8E+07 2.3E+07 4.5E+07 2.5E+06 5.6E+07 1.1E+08 GDI1 2.6E+05 1.7E+05 6.7E+05 2.6E+05 3.8E+05 1.4E+06 ADH1C 4.0E+05 8.0E+05 1.4E+06 2.6E+06 1.7E+06 4.1E+05 CD163L1 6.8E+05 5.0E+01 3.2E+03 6.7E+05 5.0E+05 8.0E+04 AKR1B10 5.8E+06 3.4E+06 8.2E+06 1.3E+07 1.3E+07 7.0E+06 LTA4H 4.4E+05 1.5E+05 5.0E+05 1.1E+06 5.3E+05 4.3E+05 GAPDH 7.9E+06 4.1E+06 1.1E+07 2.2E+07 1.3E+07 9.6E+06 CFHR1 1.8E+05 3.9E+05 7.0E+05 5.7E+04 6.9E+05 1.7E+06 IPI00914985 1.7E+05 5.0E+01 5.0E+01 5.0E+01 7.8E+04 2.6E+05 LCN2 2.6E+06 2.0E+05 4.1E+05 6.3E+06 2.1E+05 6.5E+04 PGRMC2 2.2E+05 5.0E+01 5.0E+01 2.9E+05 1.6E+05 5.0E+01 PGLS 5.6E+05 1.4E+05 7.8E+04 7.1E+05 6.4E+05 2.6E+05 SYCN 8.0E+05 1.5E+05 3.8E+03 1.5E+06 2.6E+05 1.8E+05
29 Diabetes Page 58 of 61
DNASE1 1.8E+06 2.5E+04 5.0E+01 2.2E+06 7.3E+05 9.0E+05 IPI00784773 2.4E+06 3.9E+05 4.1E+05 1.4E+05 2.5E+06 4.2E+06 ACADM 2.9E+05 1.0E+05 1.8E+05 4.8E+04 9.8E+05 2.3E+05 LGALS3 1.2E+06 7.9E+05 1.7E+06 4.7E+06 3.0E+06 5.5E+05 MSRA 1.1E+05 5.0E+01 5.0E+01 8.9E+04 1.0E+05 6.5E+04 IGHD 2.4E+05 7.4E+04 2.4E+05 5.0E+01 9.9E+05 2.9E+05 TFF2 3.3E+06 2.5E+05 8.7E+05 8.5E+06 1.0E+06 6.9E+05 HPR 3.3E+06 1.0E+06 6.9E+06 2.6E+06 1.2E+07 1.2E+07 NAPRT1 3.8E+05 9.1E+04 1.7E+05 1.0E+06 2.7E+05 3.3E+05 RNPEP 9.5E+05 4.1E+05 8.4E+05 4.2E+06 1.2E+06 4.9E+05 IPI00387024 2.4E+06 3.5E+05 1.2E+05 9.4E+04 3.5E+06 4.1E+06 GAPDHS 2.8E+05 5.0E+01 1.7E+05 7.8E+04 6.6E+05 5.0E+05 SERPINB6 1.6E+05 5.0E+01 1.1E+05 3.0E+05 3.1E+05 1.5E+05 ZG16 2.3E+06 1.7E+05 8.7E+05 1.4E+06 5.0E+06 3.1E+06 VSIG1 5.0E+01 5.0E+01 8.0E+05 2.3E+06 9.1E+02 5.0E+01 TPI1;RCTPI1 3.0E+06 5.0E+06 1.6E+06 1.2E+07 1.2E+07 4.3E+06 NPM1 1.1E+05 2.6E+05 1.4E+05 4.8E+05 9.3E+05 6.7E+04 AKR1C1 5.7E+05 7.4E+04 4.0E+05 1.7E+06 6.9E+05 8.6E+05 IPI00387109 5.0E+01 5.0E+01 4.2E+04 5.0E+01 1.3E+05 5.0E+01 EPHX1 6.2E+05 4.6E+05 9.8E+05 5.5E+06 7.7E+05 2.6E+05 ANXA1 2.2E+05 2.8E+05 6.6E+05 3.6E+06 4.7E+04 1.4E+05 FN1 2.3E+05 5.0E+01 3.9E+02 1.3E+03 3.6E+04 6.9E+05 C1RL 1.5E+05 3.7E+05 3.9E+04 1.9E+05 9.8E+05 6.5E+05 KRT2 2.9E+06 1.2E+06 5.8E+05 1.4E+06 8.3E+06 5.8E+06 LGALS4 3.6E+06 9.8E+05 1.6E+06 1.2E+07 7.1E+06 3.0E+06 LOC100130811 5.3E+04 5.0E+01 5.0E+01 5.4E+02 5.0E+01 2.0E+05 C1QC 3.1E+05 1.8E+05 1.4E+05 6.4E+04 4.3E+05 1.9E+06 MUC5B 6.5E+06 2.3E+06 4.5E+06 4.9E+07 1.4E+06 7.3E+05 FTH1 1.3E+05 5.0E+01 5.0E+01 5.0E+01 3.1E+05 1.8E+05 KRT71 5.9E+05 5.0E+01 5.0E+01 5.0E+01 1.3E+06 1.0E+06 IPI00384395 8.1E+04 5.0E+01 5.0E+01 5.0E+01 1.8E+05 1.6E+05 REG1B 5.7E+04 4.7E+04 2.0E+05 3.5E+05 7.3E+05 2.4E+05 GP2 8.6E+05 2.2E+04 5.0E+01 1.9E+05 1.8E+06 1.9E+06 RBKS 5.0E+01 5.0E+01 6.7E+04 1.7E+05 6.5E+04 5.6E+04 CPA4 8.9E+05 1.2E+05 7.1E+04 2.1E+06 1.3E+06 1.5E+06 IGHV1OR15-1 2.8E+06 1.4E+06 3.1E+05 2.5E+04 9.1E+06 1.1E+07 SERPINB1 1.7E+06 9.7E+05 2.0E+06 1.8E+07 2.7E+06 2.8E+06 IPI00383629 7.7E+05 1.5E+05 5.0E+01 9.2E+04 1.5E+06 3.0E+06 PRSS3 1.9E+06 3.3E+05 1.2E+05 7.1E+06 1.7E+06 2.8E+06 KRT1 4.0E+06 2.8E+06 2.1E+06 7.1E+06 2.3E+07 1.5E+07 APCS 5.2E+05 3.5E+05 2.9E+05 1.2E+05 1.5E+06 4.3E+06 MUC1 1.4E+05 1.1E+02 4.7E+04 7.7E+05 1.7E+05 4.4E+04 IPI00829752 1.3E+05 5.0E+01 5.0E+01 5.0E+01 2.2E+05 4.4E+05 IPI00748265 7.8E+04 5.0E+01 4.7E+04 4.7E+02 1.1E+05 5.8E+05 TAGLN2 1.1E+06 1.1E+05 2.5E+05 6.9E+06 6.8E+05 9.7E+05
30 Page 59 of 61 Diabetes
CUZD1 2.6E+06 4.7E+06 1.3E+06 1.6E+07 2.4E+07 9.6E+06 CLPS 2.9E+05 3.7E+05 2.2E+05 1.6E+06 6.1E+05 3.0E+06 PNLIPRP2 1.5E+07 5.9E+06 8.2E+05 4.3E+07 6.1E+07 3.6E+07 IL1RN 8.5E+04 1.2E+03 5.0E+01 4.6E+05 5.0E+01 9.2E+04 KRT6A 7.5E+04 1.3E+03 5.0E+01 4.9E+05 5.0E+01 5.0E+01 KLK1 9.5E+05 6.9E+05 6.8E+05 9.1E+06 5.8E+06 5.0E+05 IPI00382442 4.5E+05 5.0E+01 5.0E+01 1.6E+05 7.9E+05 2.2E+06 KRT9 2.5E+06 4.1E+05 1.5E+05 3.8E+06 8.5E+06 8.9E+06 HBE1 8.5E+05 3.6E+05 3.1E+05 2.2E+04 5.9E+06 5.1E+06 IGK@ 6.8E+04 5.0E+01 5.0E+01 5.0E+01 1.2E+05 4.0E+05 CELA2B 9.5E+05 8.5E+05 5.6E+05 1.4E+07 7.6E+06 1.3E+06 CST2 5.0E+01 1.1E+05 5.0E+01 1.1E+06 5.7E+03 5.0E+01 HEXA 6.7E+04 5.0E+01 5.0E+01 4.2E+05 1.8E+05 8.9E+04 IPI00827637 7.2E+04 5.0E+01 5.0E+01 5.0E+01 3.6E+05 4.4E+05 RNASE1 6.9E+05 1.3E+06 2.5E+06 2.9E+07 2.0E+07 2.9E+06 CPA2 7.9E+06 4.2E+06 3.8E+06 1.6E+08 2.4E+07 1.4E+07 PSMB1 1.6E+05 5.0E+01 5.0E+01 9.1E+05 7.7E+05 3.3E+05 S100A7 4.3E+04 1.4E+05 1.6E+05 4.8E+06 5.5E+04 1.1E+05 LY6D 5.0E+01 6.4E+04 5.0E+01 9.1E+05 4.0E+04 5.0E+01 CEL 1.4E+07 5.8E+06 3.6E+06 1.1E+08 2.1E+08 3.1E+07 CTRB1 1.0E+07 2.7E+06 1.5E+06 4.9E+07 1.4E+08 3.2E+07 CTRL 4.1E+05 4.2E+05 1.3E+05 6.7E+06 6.1E+06 1.9E+06 PRSS1 9.4E+06 4.2E+06 3.5E+06 1.5E+08 9.1E+07 3.3E+07 PSMA1 1.3E+05 5.0E+01 4.4E+04 1.5E+06 9.9E+05 4.6E+05 CTRL 2.0E+05 5.0E+01 5.0E+01 4.6E+05 2.3E+06 6.5E+05 CPA1 1.8E+07 4.5E+06 5.6E+06 2.9E+08 1.5E+08 4.3E+07 IPI00385254 7.4E+04 5.0E+01 5.0E+01 3.7E+04 8.4E+05 4.4E+05 KRT76 2.1E+05 5.0E+01 5.9E+04 1.1E+06 2.5E+06 1.2E+06 NME1 2.2E+05 5.0E+01 5.0E+01 2.6E+06 8.3E+05 5.8E+05 CELA3B 1.6E+06 2.4E+04 7.8E+05 3.1E+07 1.1E+07 4.4E+06 PNLIPRP1 3.1E+06 3.0E+05 4.7E+05 4.9E+07 2.2E+07 6.7E+06 FASN 1.5E+05 7.6E+04 5.5E+04 2.8E+04 1.7E+06 4.7E+06 CELA2A 2.3E+06 9.2E+05 3.9E+05 4.3E+07 3.3E+07 1.3E+07 IPI00219910 2.3E+05 8.1E+04 1.4E+05 5.1E+06 5.7E+06 6.1E+05 IPI00827829 6.4E+04 5.7E+04 3.2E+02 3.3E+06 5.0E+01 5.0E+01 PNLIP 2.8E+07 3.5E+06 7.6E+06 7.0E+08 2.7E+08 9.1E+07 PRSS2 2.6E+06 2.7E+05 6.5E+05 5.3E+07 3.6E+07 1.1E+07 CLC 5.0E+01 4.3E+04 5.0E+01 7.0E+05 3.7E+05 2.1E+05 CELA3A 4.4E+06 1.1E+06 2.2E+06 1.3E+08 7.7E+07 2.7E+07 CTRC 2.0E+06 1.2E+06 4.9E+05 4.4E+07 5.6E+07 1.2E+07 MPO 5.0E+01 7.1E+04 3.5E+04 3.4E+06 2.2E+05 5.0E+01 GP2 8.7E+04 2.3E+05 1.1E+05 1.0E+07 2.6E+06 2.2E+06 GP2 9.5E+05 8.4E+04 4.8E+05 1.4E+07 2.8E+07 1.4E+07 CPB1 4.0E+06 1.5E+06 9.4E+05 1.3E+08 8.8E+07 2.4E+07 AMY1C;AMY2A;AMY 2.1E+06 3.3E+05 5.1E+05 7.7E+07 3.3E+07 4.6E+06
31 Diabetes Page 60 of 61
1A;AMY1B AMY2B 1.0E+07 4.9E+06 3.9E+06 4.3E+08 3.2E+08 4.6E+07 SERPINI2 5.0E+01 6.7E+04 5.0E+01 3.6E+06 3.3E+06 2.9E+05 PLA2G1B 3.7E+05 4.9E+04 7.6E+04 4.0E+07 1.5E+07 3.8E+06 HMGCL 5.0E+01 5.0E+01 5.0E+01 5.0E+01 4.1E+04 5.0E+01 CD302;LY75 5.0E+01 2.7E+02 5.0E+01 1.1E+05 5.0E+01 5.0E+01 HPN 5.0E+01 8.8E+02 5.0E+01 3.4E+05 5.0E+01 5.0E+01 SCFV 5.0E+01 5.0E+01 5.0E+01 1.5E+02 5.0E+01 5.4E+04 IPI00884092 5.0E+01 5.0E+01 5.0E+01 5.0E+01 3.0E+04 3.5E+04 AZU1 5.0E+01 2.3E+03 5.0E+01 1.4E+06 3.4E+05 5.0E+01 TPM3 5.0E+01 5.0E+01 5.0E+01 5.0E+01 3.5E+04 7.6E+04 LOC100132635 5.0E+01 4.2E+03 5.0E+01 3.1E+06 6.2E+04 5.0E+01 ERO1L 5.0E+01 5.0E+01 5.0E+01 9.8E+04 6.0E+04 5.0E+01 IPI00827580 5.0E+01 5.0E+01 5.0E+01 5.0E+01 4.5E+04 1.2E+05 TTN 5.0E+01 5.0E+01 5.0E+01 4.4E+04 4.1E+04 8.1E+04 PGAM4;LOC728188 5.0E+01 5.0E+01 5.0E+01 1.3E+05 6.4E+04 5.0E+01 IPI00398220 5.0E+01 5.0E+01 5.0E+01 5.2E+02 5.0E+01 1.9E+05 BPI 5.0E+01 1.4E+03 5.0E+01 2.9E+06 5.0E+01 5.0E+01 CTRB2 5.0E+01 5.0E+01 5.0E+01 2.7E+05 1.2E+05 5.0E+01 C1QA 5.0E+01 5.0E+01 5.0E+01 3.8E+05 8.5E+04 5.0E+01 GLO1 5.0E+01 9.9E+01 5.0E+01 4.5E+05 2.2E+05 5.0E+01 AMY1C;AMY2A;AMY 5.0E+01 9.4E+02 5.0E+01 2.7E+06 1.3E+06 5.0E+01 1A;AMY1B GALM 5.0E+01 5.0E+01 5.0E+01 3.0E+05 2.9E+05 5.0E+01 GC 5.0E+01 5.0E+01 5.0E+01 5.0E+01 6.7E+05 5.0E+01 CNP 5.0E+01 5.0E+01 5.0E+01 7.9E+05 5.8E+04 5.0E+01 LCN1 5.0E+01 1.2E+02 5.0E+01 1.4E+06 5.0E+01 5.0E+01 MBL2 5.0E+01 5.0E+01 5.0E+01 1.1E+06 2.6E+04 5.0E+01 CDH1 5.0E+01 5.0E+01 5.0E+01 7.4E+05 4.2E+05 4.2E+04 IGLC1;IGLV1- 5.0E+01 5.0E+01 5.0E+01 5.0E+01 9.6E+05 6.5E+05 44;IGLV1-40;IGLV3- 21;IGLV2-11;IGLV2- 14;IGL@;IGLC2;IGLC 3 PRSS1 5.0E+01 5.0E+01 5.0E+01 2.9E+06 3.0E+05 4.2E+05 GFPT1 5.0E+01 5.0E+01 5.0E+01 4.6E+06 7.8E+05 1.0E+05
Suppl Table 3:
Map PMID
32 Page 61 of 61 Diabetes
A1 15542607
A2 11274345
A3 11606045
A4 12205041
B1 10791986
B2 16611744
B3 16122376
B4 10880430
B5 11950845
B6 16614236
B7 11368003
C1 17915332
C2 10339425
C3 12049736
C4 9164868
C5 11389839
C6 11505407
C7 Gerhard Michal: Biochemical Pathways: An Atlas of Biochemistry and
Molecular Biology
D1 15071501
D2 15716280
D3 9054461
D4 7925482
D5 Gerhard Michal: Biochemical Pathways: An Atlas of Biochemistry and
33 Diabetes Page 62 of 61
Molecular Biology
E1 12235123
34