Advanced Technology in Food Production

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

Citation:

Wakasa Y, Hirano K, Urisu A, Matsuda T, Takaiwa F. Generation of transgenic rice lines with reduced contents of multiple potential allergens using a null mutant in combination with an RNA silencing method. Plant Cell Physiol. 2011; 52 (12): 2,190-2,199.

PubMed ID: 22039121

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 create a cost-effective means to produce hypoallergenic rice with good taste for rice allergy patients and to then examine the IgE binding capacity of three transgenic rice seeds using human sera.

Inclusion Criteria:

Sera positive for IgE positive ELISA values for rice seed proteins
Informed consent.

Exclusion Criteria:

No informed consent.

Description of Study Protocol:

Recruitment
Recruited from Fujita Health University Hospital.

Design

Non-randomized crossover trial
Alpha amylase/trypsin inhibitors, alpha globulin and beta glyoxalase are regarded as major potential allergens of rice seed based on specific recognition by serum IgE from allergy patients. In order to suppress the production of these major allergens in rice grains, a mutant in the Koshihikari background lacking the 26kDa allergen was used as a host for RNA silencing.
A binary vector harboring two RNA interference gene cassettes for suppression of 14-16kDa and 33kDa allergens driven by the 13kDa and 10kDa prolamin endosperm-specific promoters, respectively, was introduced into the GbN-1 genome by agrobacterium-mediated transformation
In the most promising transgenic line, the content of three potential allergens was remarkably reduced to a very faint level without a change in seed phenotype
To evaluate some other potential allergens remaining in the established transgenic lines with reduced contents of each or all of the three major allergens, total albumin-globulin proteins were extracted from rice seeds and IgE-binding to the extracted proteins was analyzed by immuno-dot-blot analysis
Koshihikari, GbN-1 and three transgenic rice lines were used in IgE binding tests using sera from allergy patients
15 rice positive and a control (rice negative) sera were reacted with rice lines
The IgE reactivity of these subjects to seed proteins from suppressed transgenic rice was examined by immuno-dot-blot analysis.

Blinding Used
Implied with measurements.

Intervention

‘Koshihikari,’ GbN-1 and three transgenic rice lines (26/33, 14-16/26 and 14-16/26/33) with reduced allergen content.

Statistical Analysis

Paired T-tests used to analyze differences in IgE binding between rice samples
P<0.05 was significant.

Data Collection Summary:

Timing of Measurements

Seed features measured once
- Length
- Width
- Thickness
- Thousand kernel weight
- Protein content
- Amylose content
- Germination rate
- Degradation pattern of seed proteins.
IgE binding tests using patients' sera conducted once for each type of rice using dot-blotting.

Dependent Variables

IgE binding.

Independent Variables

Presence of rice allergens
‘Koshihikari,’ GbN-1 and three transgenic rice lines (26/33, 14-16/26 and 14-16/26/33) with reduced allergen content.

Control Variables

Seed features

Length
Width
Thickness
Thousand kernel weight
Protein content
Amylose content
Germination rate
Degradation pattern of seed proteins.

Description of Actual Data Sample:

Initial N: 16 rice-allergic patient seras (gender not described)
Attrition (final N): 16 rice-allergic patient seras (gender not described)
Age: Not described
Ethnicity: Not described
Other relevant demographics: Not described
Anthropometrics: Not described
Location: Japan.

Summary of Results:

Key Findings

The 15 selected serum samples containing rice specific IgE with considerable individual variability in the IgE concentration or antigen-binding affinity showed an IgE binding dot intensity five to 30 times higher than the intensity of the negative control serum binding to the albumin-globulin fraction from the control wild-type rice
Mean value of IgE binding on the serum samples gradually decreased with the stepwise deletion of the three major allergens from the rice seed protein
Mean value decreased to about 50% and 20%, respectively, of the non-transgenic control when two allergens, 33kDa plus 25kDa allergen and 14-16kDa plus 26kDa allergen, were mostly deleted
Significant reduction (P<0.001) in IgE response to 26/33kDa, 14-16/26kDa and 14-16/26/33kDa as compared to control rice
Three rice proteins were estimated to account for the majority of rice seed causative allergens recognized by IgE from patients' sera examined in the study.

Samples	Length Millimeter	Width Millimeters	Thickness Millimeter	Thousand Kernel Weight Grams	Protein Content	Amylose Content
Koshihikari	5±0.02	2.85±0.03	1.91±0.01	19.4	10.9±0.95%	16.7±2.12%
GbN-1	5.08±0.02	2.92±0.02	1.97±0.01^a	18.8	10±0.33%	15.6±1.96%
14-46/26/33	4.9±0.06^b	2.58±0.01^a,b	1.89±0.003	18.1	10.3±0.87%	17.5±0.57%

a: P<0.05 for the group of transgenic rice in comparison with the control rice
b: P<0.05 for the group of transgenic rice in comparison with the GbN-1 group.

Author Conclusion:

In the most promising transgenic line, the content of the three potential allergens was reduced to a faint level without a change in seed phenotype
The three rice proteins were estimated to account for the majority of rice seed causative allergens recognized by IgE from patients' sera examined in the study demonstrating that the sequential allergen deletion or reductions strategy works in the development of hypo-allergenic rice lines.

Funding Source:

Government:

Ministry of Agriculture, Forestry and Fisheries of Japan

Reviewer Comments:

Sera from small number of rice-allergic patients
Sample not described.

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?	Yes
	2.2.	Were criteria applied equally to all study groups?	Yes
	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?		N/A
	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?	N/A
	3.3.	Were concurrent controls or comparisons used? (Concurrent preferred over historical control or comparison groups.)	N/A
	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?	Yes
	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?	Yes
	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?	N/A
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)?	No
	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