Investigation of Genetic Markers Associated
with Non-Celiac Gluten Sensitivity
Maci Sprosty, Biology Major
Mentor: Dr. Diane A. Caporale, Department of Biology
METHODS
RESULTS
Allele (DQA1*)
05:01:01:01-01:02
02:01
03:01:01
01:04:01:01-04:02
03:03:01-03:02
01:03:01:01-03:01:02/10
05:05:01:01-05:01:03
01:02:04
03:02
Type
1
2
3
4
5
6
7
8
9
ID
Gluten Control
DQ2
6
3
DQ2-9-7
3
4
DQ8-7-4
2
DQ5-6
1
1
DQ7
2
2
DQ6-5
1
1
DQ7
1
DQ6-5
1
DQ2-4-7-8-9
(2)
1) Signed consent forms were obtained from
participating individuals
2) Saliva samples were obtained
(3)
3) Isolation of DNA using Norgen Saliva DNA
Isolation Kit
(4)
4) Amplification of 23 allele mutations by
using SSP UniTray by Life Technologies and
INTRODUCTION
(5)
Non-Celiac Gluten Sensitivity (NCGS) is a disorder whereas a
person exhibits gastrointestinal problems within 72 hours of gluten
5) Veriti Thermocycler wsas used for PCR.
ingestion. NCGS is estimated to occur in about 3-6% of the U.S.
population. This is a rough estimate since there is currently no
diagnostic tool for NCGS. However, other less common digestive
conditions, such as celiac disease (CD) and wheat allergies (WA), can
6) Gel Electrophoresis
be diagnosed with a blood. These disorders are associated with
(6)
certain types of proteins on the surface of white blood cells called
human leukocyte antigens (HLAs; Sapone et al. 2012; Tonutti and
7) Imaging using Bio-Rad’s Gel Doc EZ
Imager
Bizzaro 2014), which is the target protein for diagnosis. In contrast, in
order to diagnose NCGS) the patient has to be tested first for these
other two conditions through blood testing, to rule them out. Then
individual items from their diet are excluded, one at a time. This whole
8)DQA1 SSP UniTray Work Sheet was used
to analyze gel images.
process of elimination is costly and time consuming. Therefore, there
DISCUSSION
is a need to identify a genetic marker that can be used for direct
(7)
diagnosis.
The purpose of this study was to evaluate whether there is a genetic link to Non-Celiac Gluten Sensitivity. We screened for the HLA DQA1 alleles among individuals
The objective of this research is to find a genetic marker to help
with and without gluten sensitivity. So far, 14 participants have been genotyped and results showed a wide amount of allelic variation of this locus. Our analyses show no
diagnose this disease in a more efficient manner. Since recent
outstanding genetic patterns in the HLA DQA1 allele group between gluten sensitive and non-gluten sensitive cohorts. More participants are being screened at this time
publications suggest certain types of human leucocyte antigens
for genotype identification.
(HLAs) are associated with celiac disease, my objective focused on
identifying other DNA variants of the HLA gene, specifically the HLA
DQ alpha portion of DNA, to help find patterns associated with NonCeliac Gluten Sensitivity. This was achieved through identifying HLADQ allele types and genotypes among 20 UWSP volunteers who have
Nineteen other samples were tested but results showed more than three possible alleles for each and, therefore, could not assigned a genotype. A way to include all
ACKNOWLEDGMENTS
samples in the analysis, DNA fingerprints were generated by assigning PCR products as either present or absent for each reaction. A 0/1 matrix was generated and
phylogenetic tree was constructed. The tree reveals no distinct clades separating the two cohorts. This shows no correlation between gluten sensitivity and HLA DQA1
type. However, one cluster of 3 gluten sensitive individuals was identified. A larger sample size may reveal any significance to this finding.
This project was partly funded by the UWSP CL&S Undergraduate Education Initiative
(UEI) provided by Dr. Caporale and the UWSP Student Research Fund (URSCA).
I would like to thank Ben Piesch for his help in the lab.
When viewing the family tree, it is possible that there may be a genetic component to gluten sensitivity. Having a combination of DQ2 alleles (a heterozygous
NCGS and 20 UWSP volunteers who never experienced gluten
condition) revealed in this family may have some significance. Furthermore, since many of Generation ll did not show symptoms until after age 30, Generation lll is more
sensitivity. In addition, HLA-DQ genotypes were compared among
likely to show gluten sensitive traits once they are older.
individuals from an affected family to look for correlations between
9) Geneious program was used to produce a
phylogenetic tree.
The allele HLA DQA1 subunit binds with HLA DQB1 subunit to form the protein as seen in the top left corner. The binding between these two subunits may be the
LITERATURE CITED
their genotypes and their level of gluten sensitivity. Here I report the
cause of gluten sensitivity. To continue this research, it would be beneficial to test the individuals for their beta subunit alleles. The combined of alleles for the alpha and
results of this family pedigree and association study between HLA-DQ
beta portions can provide a label for the DQ locus. For example, DQ2 /DQ2 can describe individual l.2. This can be deduced because HLA DQA1 allele 05:01 is only seen in
Washington Post. 29, April 2011. http://www.washingtonpost.com/politics/3-years-after-
allele types and Non-Celiac Gluten Sensitivity and discuss the
the DQ2 loci. The information provided by this testing would be beneficial to identifying any genetic patterns between gluten sensitive and non-gluten sensitive individuals.
deadline-fda-still-hasn’t-defined-gluten-free/2011/04/AFRq6i8E_story_1.html
implications of these findings. This research has high relevancy, since
Non-Celiac Gluten Sensitivity is so prevalent in the U.S. population
(Leonard and Vasager, 2014).
This study would benefit greatly from a more reliable testing kit. Unfortunately, the SSP UniTray from Life Technologies had many errors with binding. These errors
resulted in no data being collected for family members I.1 and ll.7, these family members were crucial to identifying any patterns in genetics associated with gluten
sensitivity. The SSP UniTray is also discontinued as Life Technologies updates their product line. The new product may produce more reliable and distinct results.
(1) Layton, L. (2011). Three years after deadline, FDA still hasn’t defined ‘gluten free’.
(2) Leonard, M.M., & Vasagar B. (2014). US perspective on gluten-related diseases. Clin Exp
Gastroenterol. 24(7). 25-37. http://dx.doi.org/10.2147/CEG.S54567
(3) Sapone, A. et al. 2012. Spectrum of gluten-related disorders: consensus on new
nomenclature and classification. BMC Medicine 10: 13-24.