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DiaUnion status report 2020-01 - 2021-12
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DiaUnion

Status Report January 2020 - December 2021

A primary focus of DiaUnion is the Feasibility study for interregional public screening program which aims to demonstrate that DiaUnion can identify healthy children who later in life will develop any of the TRIAD diseases, type 1 diabetes (T1D), celiac disease (CD) and autoimmune thyroiditis (AIT). Two different approaches for the screening is taken:

  1. In Sweden, a total of 2000 randomly selected children from the general population in Skåne will be asked to take a screening capillary blood sample at home.
  2. In Denmark, 2000 randomly selected anonymous samples will be obtained from already established children cohorts.

Autoantibody screening of children from the general population (Sweden)

The research activities are taking place at Lund University Clinical Research Center Malmø.

The study design has been altered to include 7-8 and 14-15-year old children instead of originally 10-15 year old children, in order to better target the ages of diagnoses for the three diseases.

Following hiring of research staff (1 PhD/clinical fellow, 1 study coordinator and 2 project assistants), ethical approval and logistics setup, the feasibility study started to invite study participants in Q2 2021.  As of December 31, 2021, a total of 13852 have been invited of whom 1825 consented to participate (13.2%). Among those invited children, 1192 (65.3%) have successfully collected a capillary sample at home for screening (Table 1).

Table 1. Enrollment, DiaUnion Feasibility study

Children age 7-8 & 14-15 years

N

%

Invited

13852

100,0%

Consents received

1825

13,2%

Address unknown

186

1,3%

Consents withdrawn

42

0,3%

Received samples after informed consent

1192

8,6%

Even though the participation and successful sample rate is lower than originally expected, we expect to be able to reach the planned number of successful samples of 2000. The lack of success in taking the capillary sample at home, is most likely tied to difficulties in filling the sample tubes with the amount of blood needed for the sample. In the public screening program planned to follow the feasibility study, the amount of blood needed will be significantly reduced and the sample tubes will be replaced with novel, easier-to-use sample containers.

850 of the 1192 samples from the children have been analyzed for T1D, CD and AIT autoantibodies and 123 (14.5%) were positive for at least one autoantibody. 65 of those (7.6%) were positive for more than one autoantibody, which is important because the risk of developing one of the diseases increases significantly when positive for more than one of the disease-related autoantibodies (Table 2).

Table 2. Distribution of autoantibodies, DiaUnion Feasibility study

Children age 7-8 & 14-15 years

N

%

10-year risk of T1D

Number of general population children analyzed

850

100,0%

1,2%

10,35

Autoantibody positive children

123

14,5%

Distribution

N

Autoantibodies

Risk %

N

Positive for multiple TRIAD disease autoantibodies

3

0,4%

1

IAA, TPO, TG

15%

0,15

1

GADA, ZNT8, IAA, TPO

70%

0,7

1

GADA, IA-2A, ZNT8, TG

70%

0,7

T1D

33

3,9%

3

GADA, IA-2A, ZNT8, IAA

70%

2,1

1

GADA, IA-2A, IAA

70%

0,7

1

GADA, IA-2A

70%

0,7

2

GADA, IAA

70%

1,4

13

GADA

15%

1,95

13

IAA

15%

1,95

CD

38

4,5%

23

tTG IgG, tTG IgA

14

tTG IgG

1

tTG IgA

AIT

49

5,8%

18

TPO, TG

10

TPO

21

TG

For T1D the risk of developing the disease with one or more autoantibodies is known: With one autoantibody the 10-year risk is 15%, and with two or more autoantibodies 70%. Applying the risk profile to the autoantibody positive children, shows that 1.2% or 10 children from the cohort are expected to develop T1D within 10 years. All autoantibody positive children are referred for a clinical follow-up by a pediatrician. Clinical evaluation for any of the diseases is currently pending.

The T1D prevalence in the Øresound region is around 0.5 %, thus the 1.2% expected prevalence found in the DiaUnion feasibility screening needs to be analyzed. The hypothesis is that when reaching out to the general population to ask for participation in the DiaUnion screening feasibility study, families who are affected by autoimmune diseases are more likely to participate than those who are not. Moving forward, questions to uncover this are being asked at consent to participate.

Samples from all autoantibody positive children will be genotyped through GWAS analysis at Steno Diabetes Center Copenhagen.

Autoantibody screening of children from established school children cohorts (Denmark)

The research activities are taking place at Steno Diabetes Center Copenhagen.

The activities on the Danish side have been lagging behind due to extensive delays in the Danish ethical approval system as a result of the Covid-19 situation. The application for ethical approval was submitted in Q4 2020, it was under review for almost a year and the approval was received in September 2021. For comparison, the normal processing time is a couple of months, which was the time it took on the Swedish side.

In the meantime, research staff (1 postdoc position) has been hired, and the study design has been altered to focus on first-degree relatives (siblings; age 1-21 years) to T1D patients from the DSDB (Dansk Selskab for Børne- og Ungdomsdiabetes) cohort, in order to better target the high-risk population. 1700 samples have been identified and it is expected that close to 300 additional samples can be found. This high-risk population will be screened and analyzed for autoantibodies at Lund University Clinical Research Center Malmø.

Validation of multiplex assays against radiobinding assays for autoantibody screening

The research activities are taking place at Lund University Clinical Research Center Malmø.

In order to scale up the DiaUnion feasibility study to a full interregional public screening program, it is necessary to optimize the analysis process, which is a major bottleneck and barrier. Using new multiplex assays in an automated lab robot, it is possible to analyze for all 5 TRIAD autoantibodies automatically and at once. What previously took a week to analyze, can now be done in one day and at a lower cost. In addition the amount of blood needed for a sample is significantly reduced, lowering the risk of unsuccessful sampling.

Therefore, another aim of DiaUnion is to compare the diagnostic performance of autoantibodies analyzed in radiobinding assays with the multiplex ADAP assay technology. The ADAP technology has been expanded from 5-plex (IAA, GADA, IA-2A, ZnT8A and tTGA) to 6-plex (TPOAb added to the previous five autoantibodies). The 6-plex fulfills the criteria for efficient screening in DiaUnion study.  Lately, it has also been possible also to add assays for SARS-CoV-2 Spike 1 and Nucleocapsid Phosphoprotein virus antibodies in a 8-plex ADAP design.

Genetic screening of both cohorts

The research activities are taking place at Steno Diabetes Center Copenhagen.

In addition to the autoantibody screening, GWAS analyses are performed on all children from the Danish cohort as well as on all autoantibody positive children from the Swedish cohort. Currently, plasma (n= 1422) and DNA (n=1746) have been prepared from the Danish cohort and genotyping will be performed once the Swedish autoantibody screening has been completed. The extended genotyping is done to optimize the genetic risk score (GRS) for developing autoimmunity and subsequent disease. While the current GRS for T1D performs excellently in separating T1D individuals from non-diabetic individuals, it is not optimized to identify at-risk individuals.

Identifying novel biomarkers for type 1 diabetes

The research activities are taking place at Steno Diabetes Center Copenhagen.

We have previously in DiaUnion characterized the plasma lipidome in adolescent subjects with increased risk for T1D (PMID: 33033923). To identify lipidomic markers of potential use in screening newborns, we have just completed a comprehensive analysis of 800 lipidomic markers in cord blood of children (n=275), who later in life developed T1D and in a similar number of control cord blood samples (n=216). Analysis is ongoing. The expected outcome is that we may identify novel markers that in combination med genetic screening will have a higher predictive value in newborns and in early life.

Interactions between minor type 1 diabetes autoantibodies and their influence on disease development

The research activities are taking place at Steno Diabetes Center Copenhagen and Lund University Clinical Research Center Malmø.

In DiaUnion  we have investigated the complex interactions of minor T1D autoantibodies (NPYA and VAMP2A) and their influence on disease onset, development of diabetic ketoacidosis, the effect on the remission phase, acute T1D complications, regulation of HbA1c and long standing T1D.

NPYA has been measured using radiobinding assay for 570 T1D probands and 482 healthy siblings. Analysis of the two groups showed no significant difference in NPY levels. Likewise, no significant difference is seen when linear models are adjusted for the major autoantibodies GADA and IA2A, gender, age at onset or bicarbonate levels and the relevant SNPs at onset.

NPYA has been tested to look for an association between NPYA and c-peptide levels in the remission phase. Neither the level of or the development of c-peptide is affected by the onset levels of NPYA when there is adjusted for age at onset, gender or onset levels of the autoantibodies IAA, IA2A, GADA, ICA.

Finally, the genetic contribution to NPY-autoantibody levels  has been analyzed. This demonstrated significant association of three non-HLA loci to NPY-autoantibody levels in T1D.

In vitro transcription translation (ITT) has been done for different versions of VAMP2 in either different lengths of the protein or VAMP2 added with a methionine tail. None of them have shown a satisfactory ITT.

VAMP2 was then coupled to a bacterium protein called Maltose Binding Protein (MBP) to improve the assay. The method was tested in our cohort of T1D children and healthy siblings by measuring autoantibody IA2A and GADA coupled to MBP (MBP-IA2A MBP-GADA). Our findings suggest MBP-fusions in RBA are valid methods to analyze, especially, small, or novel antigens not possible for standardized RBA. True values of MBPA- and autoantigen-levels can be found by displacing with cold sequences of MBP or autoantigen and analyzing delta-variances. MBP-fusions to IA-2 has no conformational consequence on the protein, while, MBP-fusions to GAD65 are suggested to highlight epitope patterns specific for siblings to T1D children. The preliminary data found autoantibodies to the transmembrane part of VAMP-2 increased in T1D children.

We are currently testing MBP-VAMP2A in the remaining serum samples of the 570 T1D probands and 482 healthy siblings and will later do similar associations studies as done with NPY to the for an association to clinical parameters as bicarbonate, c-peptide and HbA1c.

Media, articles and publications

Attachment 1: Screening for type 1 diabetes, coeliac disease and thyroiditis in a general paediatric

population: the TRIAD study. Abstract for the 54th Annual Meeting of ESPGHAN 2022

Attachment 2: High-throughput multiplex assay of autoantibody biomarkers by agglutination-PCR

(ADAP) compared to radiobinding assays in type 1 diabetes and celiac disease. Poster for the Immunology of Diabetes Society (IDS) virtual meeting, November 3, 2021

Attachment 3: Multiplex agglutination-PCR (ADAP) autoantibody assays compared to radiobinding autoantibodies in type 1 diabetes and celiac disease. Submitted for publication

Attachment 4: Neuropeptide Y Autoantibodies in Type 1 Diabetes: Associations with Islet Autoantibodies, Glycemic Control, β-cell Function and Body Mass Index. Submitted for publication

Attachment 5: Genetic variants associated with neuropeptide Y autoantibody levels in newly diagnosed individuals with type 1 diabetes. Submitted for publication

The multiplex assay and automated lab robot presented on the Lund University website, February 11, 2021:

https://www.gppad.lu.se/artikel/hamilton-i-forskningens-tjanst

DiaUnion featured in Øresundsinstituttets Survey of Life Science Across the Øresund, June 22, 2021:

http://www.oresundsinstituttet.dk/wp-content/uploads/2021/06/20210622_Life_science_across_the_Oresund.pdf

The multiplex assay and automated lab robot presented in the journal Vårdfokus, October 11, 2021:

https://www.vardfokus.se/nyheter/roboten-hamilton-ger-snabb-analys-av-antikroppar/

 

Article on DiaUnion screening in Diabetes Wellness Sweden, October 18, 2021:

https://www.mynewsdesk.com/se/diabeteswellness/news/ny-studie-10-000-skaanska-barn-erbjuds-test-foer-tre-autoimmuna-sjukdomar-435346

Website: www.diaunion.org

LinkedIn: https://www.linkedin.com/company/diaunion

Twitter: https://twitter.com/DiaUnionT1D

Funding

DiaUnion is funded by: