Advanced Technology in Food Production

ATFP: Human Consumption of Plant Foods Produced Using Genetic Engineering (GE) Technologies (2015)

Citation:

Nakamura R, Satoh R, Nakamura R, Shimazaki T, Kasuga M, Yamaguchi-Shinozaki K, Kikuchi A, Watanabe KN, Teshima R. Immunoproteomic and two-dimensional difference gel electrophoresis analysis of Arabidopsis dehydration response element-binding protein 1A (DREB1A)-transgenic potato. Biol Pharm Bull. 2010; 33 (8): 1,418-1,425.

PubMed ID: 20686241

Study Design:

Non-Randomized Crossover Trial

Class:

C - Click here for explanation of classification scheme.

Quality Rating:

NEUTRAL: See Quality Criteria Checklist below.

Research Purpose:

To evaluate unintended effects of genetic modification by investigating the allergenicity of stress-tolerant transgenic potatoes expressing the Arabidopsis DREB1A (dehydration responsive element-binding protein 1A) gene, a transactivational factor against environmental stresses.

Inclusion Criteria:

Potatoes

Transgenic potatoes expressing the AtDREB1A gene, driven by the rd29A promoter and the 35S promoter
Non-transgenic potatoes were used as controls.

Patients

Positive for potato-specific immunoglobulin E (Immuno-CAP scores 3-5)
Patients without allergies were used as controls
Informed consent obtained.

Exclusion Criteria:

Not described.

Description of Study Protocol:

Recruitment

Sera from 14 potato-allergic patients were used: Four from Japan and 10 from the United States
Sera from two subjects without allergies were used as controls
Recruitment process not described.

Design
Non-randomized crossover trial.

Blinding Used
Implied with measurements.

Intervention

A comprehensive detection method (immunoblotting with sera from allergic patients) and a quantification method [two-dimensional difference gel electrophoresis (2D-DIGE)] were used to evaluate unintended effects of genetic modification on inherent allergens in potato
First, 1D- and 2D-immunoblotting using sera from patients with potato allergies was carried out. Some potato proteins that bound to immunoglobulin E (IgE) were identified by mass spectrometry [matrix assisted laser desorption/ionization-time of flight (MALDI-TOF-MS/MS)]
Finally, 2D-DIGE analysis was used to determine relative differences in the amounts of allergens and other proteins between non-transgenic and transgenic potatoes.

Statistical Analysis

After 2D-DIGE test was carried out, normalized values of protein spots from five tubers of each transgenic potato (rd29A and 35S) and five non-transgenic potatoes were calculated
Differences among these values were tested by one-way ANOVA followed by Dunnett-tests and P<0.05 was considered to indicate a significant difference.
The "ratio" denotes the mean value of a relative normalized protein spot from transgenic potatoes divided by that of a protein spot from non-transgenic potatoes
The spots showing ratios greater than two were designated as significantly different expression in transgenic vs. non-transgenic potatoes.

Data Collection Summary:

Timing of Measurements
Measurements made with sera from selected subjects.

Dependent Variables

Patterns of protein binding to IgE (by 1D- and 2D-immunoblotting)
Protein concentrations (by 2D-DIGE)
Protein identification (by mass spectrometry MALDI-TOF-MS/MS).

Independent Variables

Transgenic vs. non-transgenic potatoes
Patients with potato allergy vs. non-allergic controls.

Description of Actual Data Sample:

Initial N: 14 potato-allergic patients, two control subjects
Attrition (final N): As above
Age: Not reported
Ethnicity: Not reported
Other relevant demographics: Not reported
Anthropometrics: Not reported
Location: Japan and United States.

Summary of Results:

Key Findings

Immunoproteomic analysis using sera from potato-allergic patients revealed several IgE-binding protein spots. The patterns of protein binding were almost the same between transgenic and non-transgenic potatoes.
None of the IgE-binding protein spots showed clear qualitative differences by 2D-immunoblot analysis
The IgE-binding proteins in potato were identified as patatin precursors, a segment of serine protease inhibitor 2, proteinase inhibitor II and aspartic proteinase inhibitor
2D-DIGE analysis revealed several differences in protein expression between non-transgenic and transgenic potatoes
- Significant difference was defined as normalized fluorescence value of protein spot over the threshold ratio of two (ratio greater than two or less than two) between non-transgenic and transgenic potatoes by Dunnett test (P<0.05)
- Those showing increased expression in transgenic potatoes were identified as precursors of patatin, a major potato allergen and those showing decreased expression in transgenic potatoes were identified as lipoxygenase and glycogen (starch) synthase.
When compared with the protein expression in non-transgenic potatoes, more proteins showed altered expression in potatoes containing the rd29A promoter than in those containing the 35S promoter. This suggests that the AtDREB1A gene is expressed at lower levels in 35S potatoes than in rd29A potatoes.

Author Conclusion:

The results suggested that transgenic potatoes may express slightly higher levels of allergens, but their IgE-binding patterns were almost the same as those of control potatoes
Further research on changes in protein expressions in response to environmental factors is required to confirm whether the differences observed in this study are due to gene transfection, rather than environmental factors.

Funding Source:

Government:

Ministry of Health, Labour and Welfare; Food Safety Commission, Japan

Reviewer Comments:

Expression of patatins is affected by various factors, including tuber size, storage condition, breeding practice and environmental factors such as abiotic stress, etc.
Further analyses using different potato tubers and different patients' sera should be conducted
Small sample size in this study.

Quality Criteria Checklist: Primary Research
Relevance Questions
	1.	Would implementing the studied intervention or procedure (if found successful) result in improved outcomes for the patients/clients/population group? (Not Applicable for some epidemiological studies)	Yes
	2.	Did the authors study an outcome (dependent variable) or topic that the patients/clients/population group would care about?	Yes
	3.	Is the focus of the intervention or procedure (independent variable) or topic of study a common issue of concern to dieteticspractice?	Yes
	4.	Is the intervention or procedure feasible? (NA for some epidemiological studies)	Yes

Validity Questions
1.	Was the research question clearly stated?		Yes
	1.1.	Was (were) the specific intervention(s) or procedure(s) [independent variable(s)] identified?	Yes
	1.2.	Was (were) the outcome(s) [dependent variable(s)] clearly indicated?	Yes
	1.3.	Were the target population and setting specified?	Yes
2.	Was the selection of study subjects/patients free from bias?		???
	2.1.	Were inclusion/exclusion criteria specified (e.g., risk, point in disease progression, diagnostic or prognosis criteria), and with sufficient detail and without omitting criteria critical to the study?	???
	2.2.	Were criteria applied equally to all study groups?	???
	2.3.	Were health, demographics, and other characteristics of subjects described?	No
	2.4.	Were the subjects/patients a representative sample of the relevant population?	???
3.	Were study groups comparable?		???
	3.1.	Was the method of assigning subjects/patients to groups described and unbiased? (Method of randomization identified if RCT)	N/A
	3.2.	Were distribution of disease status, prognostic factors, and other factors (e.g., demographics) similar across study groups at baseline?	???
	3.3.	Were concurrent controls or comparisons used? (Concurrent preferred over historical control or comparison groups.)	Yes
	3.4.	If cohort study or cross-sectional study, were groups comparable on important confounding factors and/or were preexisting differences accounted for by using appropriate adjustments in statistical analysis?	N/A
	3.5.	If case control study, were potential confounding factors comparable for cases and controls? (If case series or trial with subjects serving as own control, this criterion is not applicable.)	N/A
	3.6.	If diagnostic test, was there an independent blind comparison with an appropriate reference standard (e.g., "gold standard")?	N/A
4.	Was method of handling withdrawals described?		Yes
	4.1.	Were follow-up methods described and the same for all groups?	Yes
	4.2.	Was the number, characteristics of withdrawals (i.e., dropouts, lost to follow up, attrition rate) and/or response rate (cross-sectional studies) described for each group? (Follow up goal for a strong study is 80%.)	Yes
	4.3.	Were all enrolled subjects/patients (in the original sample) accounted for?	Yes
	4.4.	Were reasons for withdrawals similar across groups?	N/A
	4.5.	If diagnostic test, was decision to perform reference test not dependent on results of test under study?	N/A
5.	Was blinding used to prevent introduction of bias?		Yes
	5.1.	In intervention study, were subjects, clinicians/practitioners, and investigators blinded to treatment group, as appropriate?	N/A
	5.2.	Were data collectors blinded for outcomes assessment? (If outcome is measured using an objective test, such as a lab value, this criterion is assumed to be met.)	Yes
	5.3.	In cohort study or cross-sectional study, were measurements of outcomes and risk factors blinded?	N/A
	5.4.	In case control study, was case definition explicit and case ascertainment not influenced by exposure status?	N/A
	5.5.	In diagnostic study, were test results blinded to patient history and other test results?	N/A
6.	Were intervention/therapeutic regimens/exposure factor or procedure and any comparison(s) described in detail? Were interveningfactors described?		Yes
	6.1.	In RCT or other intervention trial, were protocols described for all regimens studied?	Yes
	6.2.	In observational study, were interventions, study settings, and clinicians/provider described?	N/A
	6.3.	Was the intensity and duration of the intervention or exposure factor sufficient to produce a meaningful effect?	Yes
	6.4.	Was the amount of exposure and, if relevant, subject/patient compliance measured?	Yes
	6.5.	Were co-interventions (e.g., ancillary treatments, other therapies) described?	Yes
	6.6.	Were extra or unplanned treatments described?	Yes
	6.7.	Was the information for 6.4, 6.5, and 6.6 assessed the same way for all groups?	Yes
	6.8.	In diagnostic study, were details of test administration and replication sufficient?	Yes
7.	Were outcomes clearly defined and the measurements valid and reliable?		Yes
	7.1.	Were primary and secondary endpoints described and relevant to the question?	Yes
	7.2.	Were nutrition measures appropriate to question and outcomes of concern?	Yes
	7.3.	Was the period of follow-up long enough for important outcome(s) to occur?	Yes
	7.4.	Were the observations and measurements based on standard, valid, and reliable data collection instruments/tests/procedures?	Yes
	7.5.	Was the measurement of effect at an appropriate level of precision?	Yes
	7.6.	Were other factors accounted for (measured) that could affect outcomes?	Yes
	7.7.	Were the measurements conducted consistently across groups?	Yes
8.	Was the statistical analysis appropriate for the study design and type of outcome indicators?		Yes
	8.1.	Were statistical analyses adequately described and the results reported appropriately?	Yes
	8.2.	Were correct statistical tests used and assumptions of test not violated?	Yes
	8.3.	Were statistics reported with levels of significance and/or confidence intervals?	Yes
	8.4.	Was "intent to treat" analysis of outcomes done (and as appropriate, was there an analysis of outcomes for those maximally exposed or a dose-response analysis)?	N/A
	8.5.	Were adequate adjustments made for effects of confounding factors that might have affected the outcomes (e.g., multivariate analyses)?	???
	8.6.	Was clinical significance as well as statistical significance reported?	Yes
	8.7.	If negative findings, was a power calculation reported to address type 2 error?	N/A
9.	Are conclusions supported by results with biases and limitations taken into consideration?		Yes
	9.1.	Is there a discussion of findings?	Yes
	9.2.	Are biases and study limitations identified and discussed?	Yes
10.	Is bias due to study's funding or sponsorship unlikely?		Yes
	10.1.	Were sources of funding and investigators' affiliations described?	Yes
	10.2.	Was the study free from apparent conflict of interest?	Yes