investigation of genetic markers associated with …...investigation of genetic markers associated...
TRANSCRIPT
Investigation of Genetic Markers Associated with Non-Celiac Gluten Sensitivity
Maci Sprosty, Biology Major Mentor: Dr. Diane A. Caporale, Department of Biology
Non-Celiac Gluten Sensitivity (NCGS) is a disorder whereas a
person exhibits gastrointestinal problems within 72 hours of gluten
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
be diagnosed with a blood. These disorders are associated with
certain types of proteins on the surface of white blood cells called
human leukocyte antigens (HLAs; Sapone et al. 2012; Tonutti and
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
process of elimination is costly and time consuming. Therefore, there
is a need to identify a genetic marker that can be used for direct
diagnosis.
The objective of this research is to find a genetic marker to help
diagnose this disease in a more efficient manner. Since recent
publications suggest certain types of human leucocyte antigens
(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 Non-
Celiac Gluten Sensitivity. This was achieved through identifying HLA-
DQ allele types and genotypes among 20 UWSP volunteers who have
NCGS and 20 UWSP volunteers who never experienced gluten
sensitivity. In addition, HLA-DQ genotypes were compared among
individuals from an affected family to look for correlations between
their genotypes and their level of gluten sensitivity. Here I report the
results of this family pedigree and association study between HLA-DQ
allele types and Non-Celiac Gluten Sensitivity and discuss the
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).
INTRODUCTION
ACKNOWLEDGMENTS
LITERATURE CITED
DISCUSSION
RESULTS
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.
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
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
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
for genotype identification.
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
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.
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
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
likely to show gluten sensitive traits once they are older.
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
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
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
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.
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.
METHODS
1) Signed consent forms were obtained from participating individuals
4) Amplification of 23 allele mutations by using SSP UniTray by Life Technologies and
3) Isolation of DNA using Norgen Saliva DNA Isolation Kit
2) Saliva samples were obtained
6) Gel Electrophoresis
5) Veriti Thermocycler wsas used for PCR.
7) Imaging using Bio-Rad’s Gel Doc EZ Imager
8)DQA1 SSP UniTray Work Sheet was used to analyze gel images.
(2)
(3)
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(5)
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(7)
9) Geneious program was used to produce a phylogenetic tree.
(1)Layton, L. (2011). Three years after deadline, FDA still hasn’t defined ‘gluten free’.
Washington Post. 29, April 2011. http://www.washingtonpost.com/politics/3-years-after-
deadline-fda-still-hasn’t-defined-gluten-free/2011/04/AFRq6i8E_story_1.html
(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.
Allele (DQA1*) Type Gluten Control ID05:01:01:01-01:02 1 3 6 DQ2
02:01 2 4 3 DQ2-9-703:01:01 3 2 - DQ8-7-4
01:04:01:01-04:02 4 1 1 DQ5-603:03:01-03:02 5 2 - DQ7
01:03:01:01-03:01:02/10 6 2 - DQ6-505:05:01:01-05:01:03 7 1 1 DQ7
01:02:04 8 1 - DQ6-503:02 9 - 1 DQ2-4-7-8-9