Advanced Technology in Food Production

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

Citation:

Lupi R, Denery-Papini S, Rogniaux H, Lafiandra D, Rizzi C, De Carli M, Moneret-Vautrin DA, Masci S, Larre C. How much does transgenesis affect wheat allergenicity? Assessment in two GM lines over-expressing endogenous genes. J Proteomics. 2013; 80: 281-291.

PubMed ID: 23403254

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 relate the transformation of wheat lines with their potential allergenicity using patient sera
To study two types of allergies (respiratory and food) on two untransformed (wt) genotypes and their GM, which reveals that some allergens already known in respiratory allergy could be involved in children suffering from wheat food allergy
To investigate a possible change in protein accumulation caused by genetic transformation, the amounts of the major classes of proteins, albumins/globulins, gliadins and glutenins, were compared between the two GM lines and their corresponding wheat genotypes, Svevo and Bobwhite.

Inclusion Criteria:

Patients with clinically documented baker's asthma (BA) or a food allergy (FA)
Few healthy controls
Subjects signed consent forms.

Exclusion Criteria:

Not described.

Description of Study Protocol:

Recruitment: Sera were obtained from the Service of Clinical Immunology and Allergology at Epinal Hospital, France, and the University Hospital of Udine, Italy, with informed consent
Design: Non-randomized crossover trial
Blinding used: Implied with measurements.

Intervention

ELISA tests were performed to determine the concentrations of specific IgE against the albumins/globulins fractions and, for BW-wt and BW-GM only, against various gliadins and LMW-GS
A FASTA alignment was performed between new IgE-binding proteins
A threshold of amino acid identity above 35% through a window of 80 amino acids was fixed in order to determine potential cross-reactivity with known allergens.

Statistical Analysis
Not described.

Data Collection Summary:

Timing of Measurements

Measurements made on selected subjects.

Dependent Variables

IgE-bound polypeptides.

Independent Variables

Wheat kernel albumins/globulins (A/G) and gluten proteins
Allergic versus control subjects.

Description of Actual Data Sample:

Initial N: Sera were obtained from 21 patients with clinically documented baker's asthma (BA) or a food allergy (FA). Control sera were obtained from healthy volunteers.
Attrition (final N): As above
Age: Not reported
Ethnicity: Not reported
Other relevant demographics: Not reported
Anthropometrics: Not reported
Location: The Service of Clinical Immunology and Allergology at Epinal Hospital, France, and the University Hospital of Udine, Italy.

Summary of Results:

Key Findings

BW-GM line showed a marked increase in the glutenin class (45%), accompanied by a decrease of 49% and 30%for the gliadin and A/G classes, respectively.
The genetic transformation of the SV-GM line did not directly affect protein accumulation; nevertheless we observed an unexpected decrease in the Gli and Glu classes (10% and 27%, respectively) and an increase (37%) in the salt-soluble A/G class.
The large increase in the gluten protein in BW-GM due to the over-expression of an LMW-GS drove us to examine the composition of its Glu and Gli fractions using specific antibodies
The anti C-terminal LMW-GS antibodies with a broad reactivity to all LMW-GS types indicated an overall increase in this class of proteins in the BW-GM line compared to its wt line
Anti N-terminal (Mab 6×1), with a reactivity restricted to a few LMW-GS, mostly encoded by the Glu-B3 locus, showed a reduction in the amount of these LMW-GSintheGM line
IgE-binding capacity of these Gli and Glu fractions was assayed by immunoblotting with sera from four patients with food allergy to wheat
The IgE reactivity did not differ between the Gli fractions of GM and wt bread wheat lines, while that against the Glu fractions differed between the four sera
Among the glutenin sub-units of BW-wt, IgE reacted with different polypeptides in the molecular weight range of 20kDa to 80kDa while in the case of BW-GM the reactivity profile was clearly dominated by the over-expressed transgenic polypeptide
A total of 606 IgE-binding polypeptides being detected, of which 109 were spotted
Many IgE-binding polypeptides were revealed in the A/G fraction of SV-wt and SV-GM, using the three sera 68, 781 and 458
More than 82% of the IgE-binding polypeptides were common to the GM and its parent line
With reference to the same serum, only a few spots were differentially detected between the two lines, either specific to the wt line or to the GM line
In the case of serum 68, seven polypeptides were specifically detected in SV-wt, but absent from the equivalent blot on its transgenic line
In the case of sera 781 and 458, three and two specific polypeptides, respectively, were detected in SV-wt
Six and three specific polypeptides were found in the SV-GM line
A total of 27 spots in the SV-GM line and 31 in SV-wt were identified by mass spectrometry, 17 of which revealed a common identity between the two lines
A large number of IgE-binding polypeptides were revealed in the A/G fraction of BW-wt and the BW-GM line using the three sera: 68, 38 and 458
More than 90% of the IgE-binding polypeptides were common to BW-wt and the BW-GM line
Some polypeptides were specific to one genotype. In the case of sera 68 and 38, six and seven specific polypeptides were found in the BW-GM line and two and five in the wt genotype, respectively
In the case of serum 458, four specific polypeptides were detected in BW-wt and one in the BW-GM line
A toal of 27 IgE-binding polypeptides were identified in BW-wt and 24 in the BW-GM line by mass spectrometry, 17 of which revealed a common identity between the two lines
Sera from 18 allergic patients, eight suffering from BA and 10 from FA, were chosen to compare the IgE-binding capacity of the A/G fractions of the two GM wheat genotypes and their two untransformed counterparts
All these sera contained IgE specific to the A/G fraction of the wheat cv Récital, with a large range of variability among patients, from two ng to 127ng per ml
The comparison of IgE-binding levels between GM genotypes and their untransformed counterparts revealed significant differences for only two patients suffering from BA (sera 858 and 1021) and for six patients suffering from FA (sera 44, 68, 326, 403, 646 and 781)
Of these, five sera (68, 646, 781, 858 and 1021) displayed increased reactivity (of between 14% and 45%) towards the A/G fraction in the SV-GM line, in comparison with SV-wt
Concerning BW-GM vs. the wt line, small differences in specific IgE concentrations for the A/G fraction were observed for only three sera, all from patients suffering from food allergy: Sera 44 and 326 showed a decrease of 5% and 9%, respectively, whereas serum 403 showed an increase in IgE concentration of 19%.

Author Conclusion:

The originality of this paper is to relate the transformation of wheat lines with their potential allergenicity using patient sera. Such focus has never been done before in wheat and should be of interest to the researches working in this field.
Another interesting point of this paper is the study of two types of allergies (respiratory and food) on two wheat genotypes and their GM which reveals that some allergens already known in respiratory allergy could be involved in children suffering from wheat food allergy
In this paper, a classical 2D proteomic analysis and the protein identifications were performed by mass spectrometry after spot picking and in gel trypsin hydrolysis
The identified proteins were checked for their potential allergenicity. In order to have a best interpretation of protein identified in terms of potential allergens, BLAST alignments were performed by using an allergen databank (SDAP).
This allows the determination of the cross-reactivity of these identified proteins with known allergens of other species and also the prediction of a potential allergenicity
At the molecular level, a few differences were revealed between the GM lines and their counterparts: Two new IgE-binding proteins were detected for SV-GM; one already known to be allergenic; and another that has never been described as an allergen
At the quantitative level, a few differences in IgE reactivity between the GM lines and their natural counterparts were highlighted, involving five patient responses among 18
This exploratory study suggests that the transformation resulted in minor unexpected effects in relation to the potential allergenicity of GM lines, but we cannot make any conclusions about their equivalence
In order to establish whether these differences are important, they need to be compared to data on the overall variation in allergenicity within conventional crops.

Funding Source:

Industry:

AGER — Agroalimentare e Ricerca, project

Other:

University/Hospital:

Italo-French University, CIB (Italian Interuniversity Consortium for Biotechnologies), and ItPA (Italian Proteomic Association).

Reviewer Comments:

Gluten proteins are generally involved in food allergy, whereas A/G are involved in both food and respiratory allergies
This study had controls for comparison, but the number of controls was not described
Large sample size, but not a representative sample. No information was provided regarding the subjects.
It is necessary to differentiate allergies based on gluten proteins and increase in sample size based on allergies and to see the effect of allergens.

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?		No
	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?	No
	2.2.	Were criteria applied equally to all study groups?	No
	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?	No
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)	Yes
	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.)	???
	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?		N/A
	6.1.	In RCT or other intervention trial, were protocols described for all regimens studied?	N/A
	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?	No
	6.4.	Was the amount of exposure and, if relevant, subject/patient compliance measured?	N/A
	6.5.	Were co-interventions (e.g., ancillary treatments, other therapies) described?	N/A
	6.6.	Were extra or unplanned treatments described?	N/A
	6.7.	Was the information for 6.4, 6.5, and 6.6 assessed the same way for all groups?	N/A
	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?		No
	8.1.	Were statistical analyses adequately described and the results reported appropriately?	No
	8.2.	Were correct statistical tests used and assumptions of test not violated?	???
	8.3.	Were statistics reported with levels of significance and/or confidence intervals?	No
	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?	No
	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