Addressing the medical, social

and regulatory challenges of the

gluten free diet

Consultant &

inventor on patents

Dr Jason Tye-Din

MBBS, FRACP, PhD

2nd Food Allergen Management Symposium, Sydney

22nd May 2017

Gluten: a complex visco-elastic protein

• Wheat

• Barley

• Rye

• (Oats)

Gluten:• Gliadin

• Glutenin

• Great for food texture

• Bad for people with coeliac disease

Latin glutin = glue

Rise of the gluten-free diet

Reilly N, Journal of Pediatrics 2016

Rise of the gluten-free diet

Reilly N, Journal of Pediatrics 2016

“The conviction that gluten free products are generally healthier is the top motivation

for purchase” - Packaged Facts (USA based)

“The ‘health halo’ of free-from foods is a key driver of uptake” - Mintel research

• CSIRO food and health survey

• 3000 surveys distributed

• 1184 returned

• 52.9% female; 51-64 years

• 126 (10.6%) avoiding

products containing wheat

• Only 5.7% claimed a formal

medical diagnosis

• Correlated with female gender,

lesser receptiveness to

conventional medicine/greater

receptiveness to

complementary medicine

What are Australians doing?

Golley et al, Public Health Nutrition 2014

OTHER

• “It is healthier”.....No evidence

• Weight loss……..No evidence

• Autism…………..Anecdotal

• Schizophrenia….AnecdotalCoeliac disease

Strong evidence

for benefit of GFD

“Gluten sensitivity”Aka

Non-coeliac gluten sensitivity

Non-coeliac wheat sensitivity

Weak evidence

Irritable bowel syndrome

FODMAP malabsorption

Not due to gluten (!)

People purchasing Gluten free food

Wheat allergy• < 0.5%

• 80% outgrown

by teen years

Strong

evidence

Non-coeliac gluten/wheat sensitivity (NCGS/NCWS)

• Descriptive term based on patient self-report

• No diagnostic test available (Anti-gliadin antibodies are not helpful)

• Formal diagnosis: double-blind placebo-controlled gluten challenge

• In one study of 147 NCGS patients,

– 62% had not had coeliac disease properly excluded

– 24% still had persistent symptoms despite diet

– 27% were not following a proper or strict gluten free diet

Biesiekierski et al, Nutrition in Clinical Practice 2014

• NCGS/NCWS is likely to encompass a range of diagnoses

– FODMAP intolerance - Amylase trypsin inhibitors (ATIs)

– Undiagnosed coeliac disease - Wheat allergy

• NCWS patients have ↑ markers of intestinal epithelial damage (FABP-2)

and immune responses to microbial components Uhde et al, Gut 2016

• In the clinic, excluding coeliac disease & other illnesses is a priority

Protein

Water insoluble: Gluten

(gliadin & glutenins)

Water soluble: albumins, globulins

Amylase trypsin inhibitors

Starch

e.g. Fructans (a ‘FODMAP’)

Wheat grain (kernel)

Triticum aestivum(Wheat)

Hordeum vulgare(Barley)

Secale cereale(Rye)

Avena sativa(Oats)

Gluten is toxic for those with coeliac disease, but other grain

components may be important for other conditions

� � � ?

Protein

Water insoluble: Gluten

(gliadin & glutenins)

Water soluble: albumins, globulins

Amylase trypsin inhibitors

Starch

e.g. Fructans (a ‘FODMAP’)

Wheat grain (kernel)

Triticum aestivum(Wheat)

Hordeum vulgare(Barley)

Secale cereale(Rye)

Avena sativa(Oats)

Coeliac disease

Irritable bowel

Gluten is toxic for those with coeliac disease, but other grain

components may be important for other conditions

� � � ?

? Wheat sensitivity

Dietary gluten

• Wheat, rye, barley, oats

HLA-DQ2/8 and

other genes

Environmental factors

• Microbiome + others

• Elevated

morbidity

and mortality

• Impaired

quality of life QALY ~0.56-

0.66

An immune-mediated systemic disorder elicited by gluten

in genetically susceptible people

• Transglutaminase-IgA

• Villous atrophy:

• Globally 1-2%

• 1 in 70 Australians

• Rising prevalence

• Increasing in Asia

Anderson, BMC Med 2013; Lohi et al, Aliment Pharm Therapeutics 2007; Vilppula et al, BMC Gastro 2009

Dietary gluten

• Wheat, rye, barley, oats

HLA-DQ2/8 and

other genes

Environmental factors

• Microbiome + others

• Elevated

morbidity

and mortality

• Impaired

quality of life QALY ~0.56-

0.66

An immune-mediated systemic disorder elicited by gluten

in genetically susceptible people

• Transglutaminase-IgA

• Villous atrophy:

• Globally 1-2%

• 1 in 70 Australians

• Rising prevalence

• Increasing in Asia

Anderson, BMC Med 2013; Lohi et al, Aliment Pharm Therapeutics 2007; Vilppula et al, BMC Gastro 2009

Immune

§ Autoimmune thyroid disease

§ Type 1 diabetes

§ Addison’s disease

§ Pernicious anaemia (ê B12)

§ Sjogren’s syndrome

§ Alopecia (hair loss)

§ Lupus (SLE)

§ IgA deficiency

Gastrointestinal

§ Microscopic colitis

§ Pancreatic insufficiency

§ Persistently elevated liver enzymes

§ Autoimmune hepatitis

§ Primary biliary cirrhosis

§ Primary sclerosing cholangitis

Bone, joints, nerves

§ Low-trauma fracture & osteoporosis

§ Joint pain/swelling

§ Nerve problems

§ Epilepsy

§ Migraines

Other

§ Dermatitis herpetiformis

§ Dental enamel defects

§ Infertility, recurrent miscarriage

§ Depression

§ Down’s syndrome

§ Turner�s syndrome

§ Lymphoma (Non-Hodgkin’s)

Adapted from NICE clinical guideline 86. National Institute for Health and Clinical Excellence, 2009

Pathogenic CD4+ T cells targeting specific gluten

peptides drive disease

Hardy and Tye-Din, Clinical & Translational Immunology 2016

• Immunogenic gluten peptides survive digestion

• Highly immunogenic 33mer sequence:

LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF

• Deamidation by transglutaminase enhances

activation of pathogenic CD4+ T cells

• Comprehensive mapping of the T cell epitopes in

wheat, barley, rye in vivo has defined three

dominant peptidesTye-Din et al, Science Transl Med 2010

• Gluten-specific T cells help B cells produce

antibodies to select gluten peptides

Why does gluten tolerance fail in coeliac disease?

~50% of Australians have coeliac genetic (HLA)

susceptibility, but only 1.5% affected

Environmental

factors

Coeliac diseaseHealthy:

gluten tolerance HLA/non-HLAgenes

• Gluten-specific Tregs appear to have reduced suppressive

capacity in CD (Cook et al, JACI 2017)

Environmental factors and coeliac disease

• Population studies show associations with the following factors:

• INCREASED RISK

• Higher socioeconomic status and better hygiene

• Perinatal and childhood infections e.g. Rotavirus

• Medications such as antibiotics, proton pump inhibitors

• Elective Caesarean section

• DECREASED RISK

• ? Breast feeding (not supported by recent trials)

• What about the effect of gluten introduction?

Timing:

• Delayed introduction of gluten (12 months instead of 6) may delay

onset of CD but not the final number of people who develop it

• Small amounts of gluten between 4-6 months did not reduce risk(Lionetti, NEJM 2014; Vriezinga NEJM 2014)

Amount:

• Higher amount of gluten consumed before age of 2 (esp. at 12

months) may increase risk for coeliac disease development(Aronsson, Clin Gast Hep 2016)

Environmental factors and coeliac disease

• Population studies show associations with the following factors:

• INCREASED RISK

• Higher socioeconomic status and better hygiene

• Perinatal and childhood infections e.g. Rotavirus

• Medications such as antibiotics, proton pump inhibitors

• Elective Caesarean section

Dysbiosis

• Pseudomonas: increases generation of immunogenic

gluten peptides (Caminero et al, Gastroenterology 2016)

• Reovirus: compromises ability to generate oral tolerance to

gluten (Bouziat et al, Science 2017)

The gluten free diet is not an easy treatment

• Lifelong and strict

• Complex - hidden sources of gluten

• Small amounts of gluten can be harmful

• Expensive (“Pay more for less”)

• Less palatable

• Eating out can be ‘risky’

• No protection from acute symptoms

• PAL statements such as “May contain traces of gluten” are common but

can confuse patients (Zurzolo et al, Clin Exp Allergy 2017)

50 mg can cause villous atrophy

Catassi et al, 2007

The gluten free diet is not

nutritionally superior

• Can be higher in starch, sugar, fat and calories

– Fewer grains and whole grains, more refined

– High glycemic index

– Lower in protein, iron and folate

– Low in fiber (6 g/day vs 12-15 g/day vs 25-38 g/day recommended)

– Weight gain can be problematic; é fatty liver disease and metabolic

syndrome (Reilly et al, J Hepatol 2015)

– Low gluten intake inversely associated with cardiovascular events

(correlates with reduction in whole grain intake) (Lebwohl et al, BMJ 2017)

Wu et al, Br J Nutrition 2015; Kulai et al, Can J Diet Pract 2015; Jenkins et al, AJCN 1987; Berti et al, EJN 2004; Marcason et al, J Am Diet Assoc. 2011; Dickey et al, Am J Gastroenterol 2006

è Inadvertent or deliberate gluten exposure commonè Incomplete healing common (recovery 85% and full healing only 50% at 5 years on a strict diet (Newnham et al, J Gastroenterol Hepatol 2015)

A role for a urine test strip to assess

for gluten exposure?

• Gluten immunogenic peptide “GIP” assay (Biomedal/iVYDAL)

• Like a pregnancy test - results in 10 minutes

• Detects gluten immunogenic peptides based on 33mer in urine or faeces

• Highly sensitive (<100 mg ingestion)

• No association with dietary questionnaire or tTG (Comino et al, Am J Gastro 2016)

• ? May be useful to differentiate true refractory coeliac disease from non-

responsive disease due to gluten exposure

• Further research needed to define role

• In non-responsive coeliac disease, a strict diet (‘GCED’) that excludes allcereals (except rice) leads to symptom and serologic improvement

(Hollon et al, BMC Gastro 2013)

• International studies• 20-32% of sampled labelled or naturally GF products contain > 20ppm

(Thompson, J Am Diet Assoc 2010; Sharma, J Agric Food Chem 2013; Lee, J Food Prot 2014; Farage, Public Health Nutr 2016)

• Australian studies limited• NSW Food Authority report (2011): gluten present in 5% of 222 gluten-free labelled food items (3 items >20 ppm – bread mix, cereal product and a ready-to-eat meal)• Imported GF manufactured goods (N=169; 14% had detectable gluten but all <1.1ppm) (Forbes, Med J Aust 2016)

• To address members concerns when eating out, Coeliac Australia developed a GF Accreditation Program and Online Training resources

• Studies underway to assess local manufactured foods and when patients dine out in food outlets offering GF options

Barriers to success of the GFD:Is gluten contamination a real issue?

What is the optimal gluten free definition for Australia?

Aus/NZ (ANZFSC - Standard 1.2.8, Clause 1):

• Gluten free foods contain:

– No detectable gluten (limit defined by whatever can be detected)

– No oats or their products*

– No cereals containing gluten that have been malted or their products

• Low gluten foods contain no more than 200ppm (mg/kg) gluten of food

– Meant to provide choice based on tolerability and advice of doctor

• Codex Alimentarius (FAO and WHO 2008) – also adopted by US FDA

(2015) AND European commission (2009): GF < 20 ppm

The argument against a non-fixed threshold definition • As food testing becomes more sensitive it will become increasingly difficult to

produce food that can be labelled “gluten free”

• Difficult for manufacturers to guarantee no contamination; at < 5 ppm gluten

minimal environmental contaminants can be detected

• → Potential for compromised GF food availability and/or cost

• Consistency between domestic and international food standards and a global

medical therapy standard is appealing

RECOMMENDATION 10.2

“FSANZ should establish a standard defining the level of gluten in foods that can be generally tolerated by gluten-intolerant consumers, taking into account:

• the varying levels of gluten sensitivity among gluten-intolerant consumers • scientific evidence on the risks of gluten to these consumers • the costs and benefits to the Australian community.”

“A barrier for adoption of innovative technologies…”

Rigorous evidence for a safe threshold is lacking

• Uncontrolled trials and global expert opinion – supportive of 20ppm

• Only a single RDBPCFC study: (Catassi et al, 2007)– 3 month challenge of gluten 50mg/d (n=13), gluten 10mg/d (n=13) or placebo

(n=13)

– 1 patient clinical relapse (vomiting, diarrhoea) at 10mg/d - dropped-out

– After 3 months,

• Placebo: 11/13 improved villous (Vh:CrD) healing;

• 10mg/d: 6/13

• 50mg/d: 2/13 (significant deterioration compared to placebo)

• Meta-analyses: insufficient data to define a safe level (Akobeng, APT 2006; Cochrane Review 2016)

• The symptomatic, serologic and histologic response to gluten exposure

is highly heterogeneous

• A high-quality adequately powered controlled study with accurately

defined gluten challenge likely to be informative ?how to fund

Putting the issue into perspective

• These are minute amounts

– 20 ppm = 20 mg in 1,000,000 mg (1 kg)

– To consume 10mg gluten you would need to eat 0.5 kg of a food

containing 20 ppm

– To consume 50mg gluten you would need to eat 2.5 kg of a food

containing 20 ppm

– i.e. safety margin “built in”

• Not feasible to protect every patient: risk-benefit consideration

• Naturally GF foods are nutritionally better options than

refined/processed GF foods and are not affected by this issue

• Australian Consumer Law prohibits GF claims if detectable gluten

is present

• Analytical limitations of gluten testing a major consideration…

Current detection based on ELISAs to gluten sequences:

• Skerritt (w-gliadin)

• Mendez R5: rye secalin QQPFP

• G12: (QPQLPY) from immunogenic 33mer

• Sandwich versus competitive

• Different target specificities, extraction methods and reference materials

used to calibrate leads to variability between commercial assays

Potential issues:

• Glutenins not detected (immunotoxic in coeliac disease)

• Assumption of 1:1 ratio between gliadin:glutenin doesn’t always apply

• Detection of rye secalin and barley hordein – variable

• Unable to reliably detect oats avenin

• Matrix effects - poorly reported but may have a significant impact

• Effect of food processing and role of deamidation unclear

• Never clinically validated as a tool to assess food safety

Limitations of current testing

• Proteomics-based methods

– quantitiative 2D gels

– HPLC

– MS – MRM, LC-MS/MS

• Highly accurate but costly equipment and user expertise

required – not suitable for routine testing

• New developments

– Immunosensors

– Aptamers

– PCR-based methods/microarrays

– Multianalyte profiling

• Testing methods that are sensitive, accurate, practical

and reflect clinical toxicity are needed

Other approaches

What next? Some thoughts

• The challenge: balancing food safety with more accessible and

affordable GF food products

• Complex message to patients with coeliac disease – allowing

increased gluten intake goes against what doctors tell them

• In the absence of clear threshold data, “No detectable gluten” is

the safest option BUT a threshold defined by the sensitivity of the

prevailing test is not evidence-based and ultimately unworkable

• High-quality feeding study data would be informative

• Alternative claims e.g. “ultra-low gluten” may enable greater

choice for consumers

• Clinical perspective: medical follow-up to assess the impact of

any dietary changes is essential

• Food industry, clinicians, dietitians, scientists and Coeliac

Australia collaboration essential

• Coeliac Australia – Food Industry Advisory Committee

Prof Don CameronProf Katie Allen

Acknowledgements

All the volunteers with coeliac disease who take part in our research

Ms Cathy PizzeyMs Linda GarrettDr Emma Halmos

Prof Mike InouyeDr Gad AbrahamA/Prof Nicole La GrutaProf John Furness

Dr Bob Anderson

Dr Melinda Hardy Ms Amy RussellMr Adam Girardin Immunology DivisionProf Phil Hodgkin

Dr Alberto CamineroProf Elena Verdu

Dr Laura CookDr Nabila SeddekiProf Tony Kelleher