Adding DNA-test for screening of HLA-DQ2 and DQ8 to improve the early diagnosis of celiac disease at the Dutch Preventive Youth Healthcare Centres

Celiac Disease (CD) is an immune-mediated systemic disorder elicited by the
ingestion of gluten containing cereals from the normal diet, among others
wheat, rye and barley. The disease is characterized by a variable combination
of gluten-dependent clinical manifestations, CD specific antibodies, human
leukocyte antigen (HLA)-DQ2 or HLA-DQ8 haplotypes and chronic inflammation of
the small bowel[1,2]. T-cells in the lamina propria of the small bowel
recognize the gluten peptides when they are bound to the HLA class II
specificities DQ2 and/or DQ8 on antigen-presenting cells. CD is one of the most
common lifelong food- related disorders; it has a frequency of 1% in the
general population: this corresponds to 170.000 persons in the Netherlands, and
of them at least 30.000 children[3-7]. However, CD is frequently unrecognized,
partially because of its variable clinical presentations and symptoms, ranging
from malabsorption with chronic diarrhoea, poor growth in children and weight
loss, to nonspecific signs and symptoms like chronic fatigue,
osteoporosis/reduced bone mineral density, gastrointestinal symptoms or
elevated liver enzymes [5,7,8]. Unrecognized and thereby undiagnosed and
untreated disease is associated with short- and long-term complications such as
delayed puberty, neuropsychiatric disturbances, associated autoimmune disease,
miscarriages, small-for-date-births, osteoporosis, and, rarely, malignancy. CD
has a considerable health burden for society and yields extensive negative
economic consequences, thereby presenting a resource challenge for current and
future health systems [9]. Once diagnosed, the patient*s health status improves
after treatment with a gluten free diet (GFD). That timely diagnosis and
treatment of CD could be achieved by active case-finding, show the preliminary
results of the ongoing ZonMw sponsored project GLUTENSCREEN (531002001;
www.glutenscreen.nl). In this active case finding project, started at February
2019, all children aged 1-4 years who attend the Preventive Youth Health Care
Centres (YHCCs) in the region of Kennemerland are yearly assessed for 10
CD-related symptoms during their regular consultation. If a child has one or
more symptoms, the parents of the child are invited to participate. After
informed consent, a point of care test (POCT) assessing CD-specific antibodies
against tissue-transglutaminase (TGA) from a droplet of blood, is performed
onsite at the YHCCs. If the POCT is positive (TGA present), CD is highly
suspected and the child is referred to the Leiden University Medical Centre
(LUMC) for diagnosis according to the standard of care[1,2]. The preliminary
results of GLUTENSCREEN are beyond expectations: From the 14.917 children
attending the YHCCs, 5.512 (37.0%) of them had one or more CD-related symptoms.
The parents of 3.203 (58.1%) children gave informed consent for a POC-test. In
61 (1.9%) children the POC-test was positive. After additional investigations
at our hospital, CD was confirmed in 55 children (1.7% of the tested children)
(serum IgA TGA >10xULN and IgA against endomysium (EMA) positive) and ruled out
in 5 children with dubious/positive POC test: in two children HLA-DQ2/8 was
negative with negative TGA in serum (ELISA-test), in 3 children with TGA <10 x
ULN (ELISA-test) in whom small bowel biopsies showed Marsh 1 lesions. One child
still needs to be seen in the hospital (parents refused till now)
(www.glutenscreen.nl). From the parents who were invited for GLUTENSCREEN,
almost 80% is willing to participate if the POCT could be performed during the
regular visit at the YHCC. All health care professionals reported that early CD
detection by case-finding adds value to the preventive care they offer at the
YHCCs. These preliminary results of our active CD case-finding project at the
Preventive YHCCs in the Netherlands illustrate that early detection of CD at
the Preventive YHC is feasible and well-accepted by parents and health care
professionals. In GLUTENSCREEN all symptomatic children will be annually tested
for CD at the Preventive YHCCs till the age of 4 years. In the region
Kennemerland, GLUTENSCREEN has already been implemented in the regular care and
POCT will be performed during consultation. However, the development of CD
requires genetic susceptibility, present in 40% of the general population.
Since the disease almost exclusively occurs in individuals with the human
leukocyte antigen (HLA)-DQ2 and/or HLA- DQ8 haplotypes, codified by chromosome
6. Because of the high negative predictive value of HLA-typing for development
of CD, repeated CD testing will be unnecessary in HLA-DQ2/DQ8 negative
individuals (60% of the general population). Currently, HLA-typing is not a
part of GLUTENSCREEN because current technique presents important drawbacks in
settings without the availability of a laboratory, like the Preventive Care
setting as the YHCCs, since it requires DNA extraction. Material for DNA
extraction is usually obtained from whole blood (minimum quantity 4-5 ml) or
from other cells, such as buccal mucosa. However, venipunctures are not
feasible at the YHCCs and buccal mucosa DNA extraction in children is
time-consuming. We here propose to develop a novel test for DNA isolation for
HLA typing extracted from the dried blood spots obtained from the POCT at the
Preventive YHCCs for early detection of CD. Other projects have shown that the
usage of real-time polymerase chain reaction (RT-PCR) on DNA acquired from
dried blood samples is successful and can easily be applied to HLA-DQ2/DQ8
typing in this setting[10]. Adding HLA-DQ2/8 typing to the case finding
strategy for CD at the Preventive YHCC is innovative since HLA-typing for CD
has not previously been done in dried blood spots in the proposed setting.
Furthermore, embedding this technique represents a novel approach to active
case-finding of CD and consequently will improve secondary prevention of CD by
early diagnosis at the Preventive YHCCs. The outcome of the proposed study will
have impact on the active case finding procedure of CD at the YHCCs. Repeated
testing for CD could be omitted in children tested HLA-DQ2/8 negative, this
reflects to 60% of the targeted population. This study will result in: 1. More
efficient and decrease of the burden of the HLA-DQ2/8 negative children: less
children have to be tested. 2. Clarity of the acceptability of HLA typing for
CD in parents from young children. 3. Costs saving by reducing unnecessary
follow up of children. To embed this technique in the case finding setting at
the YHCCs, the test will be offered to a significant part of the general Dutch
population between 0-4 years old, since more than 95% of the general population
visit the YHCC.

Primary Objective:
1. To validate the HLA-DQ typing in blood taken by a fingerprick; to make it
feasible in the regular Preventive YHCCs organization.
2. To establish the feasibility of HLA-DQ2/8 typing in the active case-finding
at the Preventive YHCCs in the region Kennemerland.
3. To establish the acceptability and impact of genetic testing (of HLA-DQ2/8)
as part of active case-finding of CD by the parents of the children attending
the Preventive YHCCs and by the health care providers at these centers

Secondary Objective(s):
To evaluate the cost-effectiveness of genetic testing of HLA-DQ2/8 as part of
the active case-finding approach

Phase 1: Validation will be done on the outpatient clinic in 50 consecutive
children (1-18years) who attend the LUMC dept. of Pediatrics because of
suspected CD in whom traditional HLA-typing is part of their standard of care.

Phase 2 (implement the HLA-test): Prospective intervention cohort study.
All parents of) symptomatic children, 1-4 years of age, who visit the
Preventive YHCC in the region of Kennemerland for a regular visit will be
invited for this study.

Acceptability and cost-effectiveness of adding the novel test to the standard
of care will also be assessed by standardized questionnaires.
As no validated/published questionnaires are available for the impact of
HLA-testing in the general population, questionnaires will be developed
together with the medical ethicist.

The duration of this study is 18 months:
The first 6 months will be used to arrange the organizational and
administrative procedures.

LUMC:
The project will be submit for approval to the Medical Ethical Committee
Leiden- Den Haag-Delft (METC-LDD)

Validation of the HLA-DQ2/8 typing in DNA isolated from dried blood spots
obtained from one droplet of blood from 50 children attending the LUMC dept. of
Pediatrics. Every month at least 15 children with (suspicion) of CD visit our
outpatient clinic at the LUMC. Assuming a conservative participation of 60% (54
children) will be sufficient to complete the work. Sensitivity and specificity
of the innovative method for HLA-DQ2/8 typing win dried blood spots in children
will be calculated.

Preventive YHCCs:
• Preparation of the work at the Preventive YHCCs, including:
o Information to the health care workers about the study;
o Sending information letters to the parents of the children involved;
o Provision of the material to the YHCC locations (filter paper with
identification number, shipping material);
o Arrangement of secure mail (*zorgmail*) to communicate the results between
laboratory and YHCCs;
o Adaptation of the YHCC medical file to allow entering the HLA-DQ2/8 results
and to make a notice that repeated CD testing is not needed;
o Building a CASTOR database for storing data and analysis of the results

In month 6-12 of the study dried blood spots will be obtained at the YHCCs
Kennemerland for HLA-DQ typing as part of early detection of CD. Collection of
HLA results into the YHCCs files and collection of the information concerning
acceptability by parents and health care providers.

During the last 6 months (month 12-18): Analysis and interpretation of the
results will be done. Preparation of a scientific paper to be submitted for
publication to an (international) peer-reviewed scientific journal.

Phase 1 (Development and Validation of HLA- DQ2/8 typing using RT-PCR in DNA
isolated from dried blood spots obtained from one droplet of blood) From 50
children attending the LUMC dept. of Pediatrics because of suspected CD in whom
traditional HLA-typing is part of their standard of care or from children with
diagnosed CD in whom their HLA typing is already known. The parents of) these
children will be asked to participate in the study. Prior to the regular
consultation, (parents of) children will receive study information letter along
with an informed consent form. After informed consent a blood droplet will be
obtained from a fingerprick which will be done after taken blood for usual
medical care (blood withdrawal by venipuncture) and deposited on filter paper
S&S 903* (Schleicher and Schuell). The dried blood spots require an additional
fingerprick to compare the collection method with that performed at the
Preventive YHCCs. DNA will be isolated using the QIAamp method designated for
this purpose from dried blood spots (Qiagen). Phase 2 (Implementation of
HLA-DQ2/8 typing on the Preventive YHCCs) All parents of symptomatic children,
1-4 years of age, who visit the Preventive YHCC in the region of Kennemerland,
will be asked to participate in this study. Prior to the visit for POCT,
(parents of) children will receive study information letter along with an
informed consent form. After informed consent, an extra droplet of blood will
be collected for the novel HLA-DQ2/8 typing by the finger prick when performing
the POCT for TGA determination as done in GLUTENSCREEN.

Benefits and risks assessment, group relatedness

The overall burden and risk for participation in this study can be considered
minimal.
Phase 1: no extra risk for participants regarding the venipuncture since it is
part of the standard care children receive when CD is suspected. Risks of
venipuncture are: limited pain from puncture and/or hematoma. An additional
finger prick will be performed to obtain a blood droplet
Phase 2: No extra finger prick is necessary for HLA-DQ typing since the
children already participate in GLUTENSCREEN and get a finger prick for
POC-test for TGA determination. Only an extra droplet of blood will be obtained
for HLA-DQ-typing. So the risks are negligible. Related to a finger prick:
limited pain from puncture, risk of skin infection negligible, risk of hematoma
negligible.

HLA-DQ2/8-positive results may cause temporary negative feelings among parents,
but our group has shown that genetic testing for CD did not expected to affect
perceived health and health-related quality of life of children[19]. Evaluating
the acceptance of genetic testing of HLA-DQ2/8 in children by questionnaires
which will be developed in cooperation with the medical ethicist, is one of the
study objectives. These results will give more insights in the usefulness to
risk ratio of genetic testing for CD predisposition in children.

All things considered, the results of the study will increase the knowledge on
the impact on the parents, children and Preventive YHCCs of the results of
genetic testing for CD-predisposition in symptomatic children. The
implementation of the results will prevent unnecessary repeated testing for CD
in approx. 60% of the children tested for early case-finding at the Preventive
YHCCs. In addition, it will provide relief to their parents that their children
will never develop CD, not at the moment of the test or during the rest of
their lives.