U.S. patent application number 14/990504 was filed with the patent office on 2016-07-07 for type 1 diabetes biomarkers.
This patent application is currently assigned to Arizona Board of Regents on behalf of Arizona State University. The applicant listed for this patent is Mark A. Atkinson, Xiaofang Bian, Joshua LaBaer, Ji Qiu, Desmond A. Schatz, Clive H. Wasserfall. Invention is credited to Mark A. Atkinson, Xiaofang Bian, Joshua LaBaer, Ji Qiu, Desmond A. Schatz, Clive H. Wasserfall.
Application Number | 20160195546 14/990504 |
Document ID | / |
Family ID | 56286356 |
Filed Date | 2016-07-07 |
United States Patent
Application |
20160195546 |
Kind Code |
A1 |
LaBaer; Joshua ; et
al. |
July 7, 2016 |
Type 1 Diabetes Biomarkers
Abstract
Type 1 diabetes (T1D) patients make antibodies to self-proteins
that are potential biomarkers for early detection and risk
prediction. We have identified seventeen antigens as biomarkers for
early diagnosis and risk prediction of T1D, including the antigens
MLH1, MTIF3, PPIL2, NUP50, TOX4, FIGN, C9orf142, ZNF280D, HES1,
QRFPR, CTRC, SNX6, SYTL4, ELA2A, IGRP, PAX6, and HMGN3.
Inventors: |
LaBaer; Joshua; (Chandler,
AZ) ; Qiu; Ji; (Chandler, AZ) ; Bian;
Xiaofang; (Mesa, AZ) ; Schatz; Desmond A.;
(Gainesville, FL) ; Wasserfall; Clive H.;
(Gainesville, FL) ; Atkinson; Mark A.;
(Gainesville, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LaBaer; Joshua
Qiu; Ji
Bian; Xiaofang
Schatz; Desmond A.
Wasserfall; Clive H.
Atkinson; Mark A. |
Chandler
Chandler
Mesa
Gainesville
Gainesville
Gainesville |
AZ
AZ
AZ
FL
FL
FL |
US
US
US
US
US
US |
|
|
Assignee: |
Arizona Board of Regents on behalf
of Arizona State University
Scottsdale
AZ
University of Florida Research Foundation, Incorporated
Gainesville
FL
|
Family ID: |
56286356 |
Appl. No.: |
14/990504 |
Filed: |
January 7, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62100775 |
Jan 7, 2015 |
|
|
|
Current U.S.
Class: |
506/9 ; 435/7.4;
435/7.92; 436/501 |
Current CPC
Class: |
G01N 2800/50 20130101;
G01N 33/6893 20130101; G01N 2800/042 20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Claims
1. A method of diagnosing Type 1 diabetes onset, comprising the
step of contacting an antibody-containing fluid sample from a
subject with one or more of antigens selected from the group
consisting of MLH1, MTIF3, PPIL2, NUP50, TOX4, FIGN, C9orf142,
ZNF280D, HES1, QRFPR, CTRC, SNX6, SYTL4, ELA2A, IGRP, PAX6, and
HMGN3, wherein detection of antibodies with a suitable detection
agent to one or more of these antigens indicates a diagnosis of
Type 1 diabetes onset in comparison to a healthy control
sample.
2. A method of screening for a risk factor associated with Type 1
diabetes onset, comprising the step of contacting an
antibody-containing fluid sample from a subject with one or more
antigens selected from the group consisting of MLH1, MTIF3, PPIL2,
NUP50, TOX4, FIGN, C9orf142, ZNF280D, HES1, QRFPR, CTRC, SNX6,
SYTL4, ELA2A, IGRP, PAX6, and HMGN3, wherein detection of
antibodies with a suitable detection agent to one or more of these
antigens indicates an elevated risk of Type 1 diabetes onset in
comparison to a healthy control sample.
3. A kit for assessing the presence of antigens for Type 1
diabetes, comprising an antigen selected from the following: MLH1,
MTIF3, PPIL2, NUP50, TOX4, FIGN, C9orf142, ZNF280D, HES1, QRFPR,
CTRC, SNX6, SYTL4, ELA2A, IGRP, PAX6, and HMGN to profile
serological antibodies, and a suitable detection agent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 62/100,775, filed Jan. 7, 2015, the entire contents
of which are incorporated herein in their entirety by
reference.
FIELD OF THE INVENTION
[0002] This disclosure relates to biomarkers for the prediction of
Type 1 diabetes (T1D) onset and for diagnosing T1D.
BACKGROUND OF THE INVENTION
[0003] T1D is one of the most common juvenile autoimmune diseases.
It is characterized by progressive autoimmune destruction of
pancreatic beta cells. The incidence of T1D is increasing
worldwide. T1D patients are dependent on lifelong exogenous insulin
but this is not a cure and in the long term there are serious
co-morbidities. This leads to both personal and societal burdens in
terms of financial and quality of life indicators. At the time of
T1D diagnosis, it is thought that potentially 70%-90% of pancreatic
beta cells have been destroyed. Therefore early diagnostic and
prognostic markers of T1D prior to symptomatic disease onset will
be of great value in identifying individuals that could benefit
from intervention protocols while significant beta cell function
still exists.
[0004] Prevention of T1D will only be possible if individuals with
high risk for progression to T1D can be identified. The incidence
of T1D in general population is around 22/100,000 in the US. The
majority of T1D cases are diagnosed in non-relatives with 85% of
new T1D cases occurring in individuals with no known family
history. Thus, biomarkers are needed to improve our prediction
models and enable the selection of subjects with, for example, high
5-year risk of disease onset. Such markers could be deployed
immediately to identify high-risk subjects for intervention trials.
Additionally in differentiating type 1 diabetes from other forms of
diabetes mellitus autoantibodies are helpful and yet there are
still some individuals with T1D that are negative for the current
known autoantibodies, thus the discovery of additional
autoantibodies aids in the differential diagnosis of T1D.
SUMMARY OF THE INVENTION
[0005] Identifying markers that present prior to the development of
our currently used autoantibodies (AAbs) could improve the risk
prediction models. Thus, the embodiments disclosed herein relate to
the identification of AAb biomarkers in T1D so as to increase the
sensitivity of detection in T1D patients and improve the T1D risk
prediction model.
[0006] All references disclosed throughout are hereby incorporated
herein in their entirety.
[0007] In one embodiment, we used a novel protein microarray
technology termed "Nucleic Acid Programmable Protein Array" (NAPPA)
(see, e.g., EP 1360490B1). This innovative protein microarray
format avoids the need to express and purify the proteins by
substituting the printing of full length cDNAs on the arrays.
Proteins corresponding to the cDNAs are produced in situ as needed
at the time of the assay by in vitro transcription and translation
(IVTT)-coupled cell lysates. The cDNAs are configured to append a
common epitope tag to all of the proteins on their C-termini so
that they can be captured by a high-affinity capture reagent that
is immobilized along with the cDNA.
[0008] In another embodiment, a method of diagnosing Type 1
diabetes onset includes the step of contacting an
antibody-containing fluid sample from a subject with one or more of
antigens selected from the group consisting of MLH1, MTIF3, PPIL2,
NUP50, TOX4, FIGN, C9orf142, ZNF280D, HES1, QRFPR, CTRC, SNX6,
SYTL4, ELA2A, IGRP, PAX6, and HMGN3, wherein detection of
antibodies with a suitable detection agent to one or more of these
antigens indicates a diagnosis of Type 1 diabetes onset in
comparison to a healthy control sample.
[0009] In a further embodiment, a method of screening for a risk
factor associated with Type 1 diabetes onset includes the step of
contacting an antibody-containing fluid sample from a subject with
one or more antigens selected from the group consisting of MLH1,
MTIF3, PPIL2, NUP50, TOX4, FIGN, C9orf142, ZNF280D, HES1, QRFPR,
CTRC, SNX6, SYTL4, ELA2A, IGRP, PAX6, and HMGN3, wherein detection
of antibodies with a suitable detection agent to one or more of
these antigens indicates an elevated risk of Type 1 diabetes onset
in comparison to a healthy control sample.
[0010] These and other aspects of the embodiments disclosed herein
will be apparent upon reference to the following disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 depicts jitter plots of representative autoantigens
from screening and knowledge based approaches (T1D stands for
new-onset T1D patients; HC stands for healthy controls).
DETAILED DESCRIPTION OF THE INVENTION
[0012] The embodiments disclosed herein relate to 17 antigens that
have been identified as biomarkers for early detection and risk
prediction of T1D. These antigens are: MLH1, MTIF3, PPIL2, NUP50,
TOX4, FIGN, C9orf142, ZNF280D, HES1, QRFPR, CTRC, SNX6, SYTL4,
ELA2A, IGRP, PAX6, and HMGN3.
[0013] In general, two approaches were used to discover the
disclosed antigens: a screen based approach and a knowledge based
approach. To profile the serological antibody response, 40 T1D
patients and 40 age/gender matched healthy controls were screened
against 10,000 human proteins across 5 NAPPA array sets.
[0014] 40 antigens were chosen for enzyme-linked immunosorbent
assay (ELISA) verification on the same sample set. 19 antigens
verified by ELISA were processed to the validation stage with 60
T1D patients and 60 healthy controls. In the knowledge based
approach, 126 pancreas enriched genes were selected from literature
mining and bioinformatics analysis and measured for their
sero-reactivity among 46 T1D patients and 46 healthy controls. 15
antigens were chosen for validation in 50 T1D patients and 50
healthy controls.
[0015] Kits for assessing the presence of antigens for Type 1
diabetes are also contemplated. An exemplary kit includes an
antigen selected from MLH1, MTIF3, PPIL2, NUP50, TOX4, FIGN,
C9orf142, ZNF280D, HES1, QRFPR, CTRC, SNX6, SYTL4, ELA2A, IGRP,
PAX6, and HMGN3 to test serological antibodies, as well as a
suitable detection agent (e.g., a labeled secondary antibody).
Example
[0016] Sera from T1D patients contain AAbs to human self-proteins.
Thus, the sero-reactivity to 10,000 human proteins with sera from
T1D patients and measured bound IgG. We scaled down the candidate
number for validation in an independent sample set. In the
knowledge based approach, we performed ELISA on 126 pancreas
enriched genes and validate the candidates in an independent sample
set. Taken together, 17 potential autoantigens were identified with
sensitivities ranging from 10-27% at 95% specificity (Table 1).
[0017] Rapid antigenic protein in situ display (Rapid) ELISA was
performed to confirm the sensitivities of autoantibodies biomakers.
96-well ELISA plates (Corning, Me.) were coated with 10 ng/mL
anti-glutathione S-transferase (GST) antibody (GE Healthcare, Pa.)
in coating buffer (0.5 M carbonate bicarbonate buffer, pH 9.6)
overnight at 4.degree. C. On the next day, coated plates were
washed 3 times with PBST and blocked with 5% milk-PBST (0.2% Tween)
for 1.5 hrs at room temperature (RT).
[0018] Meanwhile, 40 ng/.mu.L plasmids encoding candidate
autoantigens were expressed in the human Hela cell-lysate based
expression system at 30.degree. C. for 1.5 hrs. After expression,
candidate autoantigens were diluted in milk-PBST and captured in
ELISA plates at 500 rpm for 1 h at RT. Plates were washed 5 times
with PBST and incubated with diluted serum samples at 500 rpm for 1
h at RT. Then plates were washed again and incubated with HRP
labeled anti-human secondary antibody (Jackson ImmunoResearch
Laboratories, Pa.) for 1 h.
[0019] Finally, the plates were washed and incubated by 1-Step
Ultra TMB--ELISA Substrate (Thermo scientific, Ill.) for detection
and sulfuric acid to stop the reaction. OD450 was measured by
Envision Multilabel Reader (Perkin Elmer, Mass.). Expression of
candidate autoantigens was confirmed by mouse monoclonal anti-GST
primary antibody and HRP labeled anti-mouse secondary antibody
detection on the same plate. Relative absorbance was obtained by
using the raw ELISA data dividing by the medium signal of each
sample across all the antigens tested on the same day. The
sensitivities for each antigen were determined at 95% specificity
in comparison to a healthy control sample.
[0020] Prior work indicated that there are four known AAb
biomarkers identified in T1D. The 5-year risk for T1D is 20-25% for
subjects with one AAb, 50-60% for subjects with two AAbs, near 70%
for subjects with three AAbs and 80% for those with four AAbs.
Additional AAb biomarkers will help to improve the risk prediction
in the general population. Thus, for example, the presence of
autoantibodies to the antigen proteins as disclosed herein could be
tested by immuno-assays. The presence of one or more autoantibodies
disclosed herein could be used as prediction of T1D onset.
[0021] The embodiments and example described above are not intended
to be limiting.
TABLE-US-00001 TABLE 1 Discovery and Validation Statistics for 17
T1D biomarkers Discovery Validation All Sensi- Specif- Sensi-
Specif- Sensi- Specif- Antigen tivity icity tivity icity tivity
icity MLH1 0.15 0.95 0.33 0.95 0.27 0.95 MTIF3 0.15 0.95 0.20 0.95
0.25 0.95 QRFPR 0.13 0.95 0.06 0.95 0.20 0.95 PPIL2 0.20 0.95 0.18
0.95 0.19 0.95 NUP50 0.15 0.95 0.17 0.95 0.16 0.95 CTRC 0.17 0.95
0.08 0.95 0.15 0.95 SNX6 0.13 0.95 0.04 0.95 0.15 0.95 TOX4 0.20
0.95 0.12 0.95 0.13 0.95 FIGN 0.13 0.95 0.12 0.95 0.13 0.95 SYTL4
0.20 0.95 0.02 0.95 0.13 0.95 ELA2A 0.11 0.95 0.04 0.95 0.13 0.95
C9orf142 0.18 0.95 0.05 0.95 0.11 0.95 ZNF280D 0.13 0.95 0.08 0.95
0.11 0.95 HES1 0.10 0.95 0.15 0.95 0.11 0.95 IGRP 0.17 0.95 0.02
0.95 0.11 0.95 PAX6 0.15 0.95 0.08 0.95 0.11 0.95 HMGN3 0.28 0.95
0.08 0.95 0.10 0.95
* * * * *