Celiac disease – Genetic testing and clinical utility

Celiac disease is a digestive disease that damages the small intestine and interferes with absorption of nutrients from food. People who have celiac disease cannot tolerate gluten, a protein in wheat, rye, and barley. Gluten is found mainly in foods but may also be found in everyday products such as medicines, vitamins, and lip balms. When people with celiac disease eat foods or use products containing gluten, their immune system responds by damaging or destroying villi—the tiny, fingerlike protrusions lining the small intestine. Villi normally allow nutrients from food to be absorbed through the walls of the small intestine into the bloodstream. Without healthy villi, a person becomes malnourished, no matter how much food one eats. Celiac disease is both a disease of malabsorption—meaning nutrients are not absorbed properly—and an abnormal immune reaction to gluten. Celiac disease is also known as celiac sprue, nontropical sprue, and gluten-sensitive enteropathy. Celiac disease is genetic, meaning it runs in families. Sometimes the disease is triggered—or becomes active for the first time—after surgery, pregnancy, childbirth, viral infection, or severe emotional stress (NIDDK).

CD is caused by a reaction to gliadin, a prolamin (gluten protein) found in wheat, and similar proteins found in the crops of the tribe Triticeae (which includes other cultivars such as barley and rye). Upon exposure to gliadin, and certain other prolamins, the enzyme tissue transglutaminase modifies the protein, and the immune system cross-reacts with the small-bowel tissue, causing an inflammatory reaction. That leads to a truncating of the villi lining the small intestine (called villous atrophy). This interferes with the absorption of nutrients, because the intestinal villi are responsible for absorption. The only known effective treatment is a lifelong gluten-free diet.

From a genetic testing point of view CD is an interesting case, the majority (>97%) of people with CD have either HLA DQ2, DQ8 or both and the number of alleles also influences the risk of developing the disease. Having said that, roughly 30% of the population carry one or more of these alleles but the prevalence of CD is only around 1-2%, so the HLA alleles are necessary, but not sufficient, for the development of celiac disease. They are not particularly predictive either (although homozygous DQ2 and DQ2/DQ8 have a much higher risk, of the order of 1/7-1/10 – Megiorni et al, 2009) so is there any use in testing for these alleles in asymptomatic individuals?  The interesting thing is that a genetic test based on HLA alleles has an almost perfect negative predictive value, if the HLA alleles are not there then the chances of developing, or of any symptoms being due to, CD are minimal, move on to the next candidate for the diagnosis. There are also arguments in favour of testing based on some interesting facts about CD:

1. The majority of people with CD don’t even know that they have it, only about 10-20% of cases have been diagnosed, the rest remain undiagnosed for a variety of reasons, mainly because symptoms are not yet so severe that they lead to diagnostic testing. The diagnosis itself is not so easy either, the full diagnosis requires a biopsy of the intestine and histological examination of the villi to establish that they are damaged (JAMA commentary).

2. The biopsy is the final diagnosis if the individual tests positive for anti-TG (transglutaminase) and/or anti-endomysium antibodies. In most countries (at least in Europe) the biopsy is required to be positive before there is any reimbursement of gluten free foods, which are quite expensive. This is not really a good situation, often it is the case that gluten sensitivity is suspected and the sources are eliminated leading to some recovery – at this point, if the patient wants reimbursement he/she has to become ill again, and not trivially since it involves actual physical damage to the small intestine.

3. CD is on the increase, in the USA levels are 4 fold higher than 50 years ago, and this is not because of better diagnosis as the samples were compared to actual stored blood samples, the data reflect a true increase in the prevalence of the disease (Rubio-Tapia, et. Al, 2009)

4. Approx 50% of people with type 1 diabetes showed some immunological reaction to gluten and other wheat proteins and about 10% are actually diagnosed as CD (Mojibian, 2009)

5. CD sufferers have higher mortality, as described in a recent study of >40,000 individuals (Ludvigsson, 2009), but the surprising finding was that even higher mortality was seen in the antibody positive but biopsy negative groups:

 

	HR for increased mortality
Celiac disease	1.39
Inflammation (no villous atrophy)	1.72
Latent Celiac (antibodies but normal mucosa)	1.35

In Sweden only those with celiac disease are treated with a gluten free diet while very few with inflammation or latent disease are. The hypothesis is that the continued consumption has health risks. Note that the study looked at mortality rates but of course this is a marker for overall poorer long term health and increased incidence of chronic disease. Many European countries will only reimburse gluten free foods for those with full celiac disease – these data make the diagnostic situation even less acceptable.

6. CD may be preventable? There is evidence that infant feeding can affect onset of CD. Interesting data from the Swedish epidemic when CD levels went up 3-4-fold during 1984-1996 whereas there was no change in neighbouring countries. The rise in CD coincided with a change in the instructions given to mothers (for which compliance is >90% in Sweden), leading to abrupt gluten introduction and cessation of breast feeding. In 1996 the instructions were changed to encourage longer breast feeding and to introduce gluten gradually between months 4-6 and while still breast feeding – the CD rate dropped 4-fold in the ensuing years (Olsson, 2008; Myléus, 2009).

7. A study by Norris et al showed that feeding between 4-6 months was linked to the lowest risk of developing CD (Norris 2005).

The combined evidence lead to the ESPGHAN (European Society for Pediatric Gastroenterology, Hepatology, and Nutrition, Agostoni et al 2008) to issue a statement advising avoidance of early (<4 months) and late (>6 months) introduction of gluten, and to do so gradually while still breast feeding. Although the evidence is not from clinical trials, babies have to be fed and a choice needs to be made, in the circumstances the evidence points to this being the best advice (there are some clinical trials in progress or recruiting but they seem to be comparing introduction at 6 months and 12 months, except for 1 Finnish trial which does not specify - clinicaltrials.gov). As always, clinical trials and nutrition is a difficult subject – given the evidence would you want your child to be in the late introduction arm?

Arguments for genetic testing: In newborns it would be helpful to identify the genetically predisposed so that the correct feeding advice can be given (it’s not just celiac disease but other autoimmune diseases as well, with shared HLA alleles). Why test though, why not give the same advice to all? The feeding program needs to be carefully followed and not all populations are as compliant as the Swedish (certainly not the Italians!) so it makes sense to target just the at risk groups, and if we don’t start somewhere we’ll never get to personalised medicine!

Are some people squeamish about the idea of genetically testing newborns for disease risk? It shouldn’t be the case here, there is a clear prevention advantage and in any case, if it were proposed to serologically HLA type them then I bet there would not be protests – it’s genetic exceptionalism…

For older children and adults – at least it can be useful in relatives of CD patients, excluding those who are not predisposed and identifying those who should be screened for auto-antibodies and then the biopsy if necessary. As a general screen? Maybe not an economical test yet for the state or insurers to pay for but it could be encouraged, the statistic that only 10-20% of CD sufferers are diagnosed is highly significant. If the true numbers were correctly diagnosed, and also if the bar was lowered to include antibody positives then all of a sudden there would be 10-20 fold more gluten sensitive individuals and this would have a positive effect on the gluten free food market, prices would drop, choice would increase and the lot of the CD suffer would improve. After all it’s not really necessary for it to be a disease, “just” avoid the gluten. Unfortunately it’s not so easy to do so, gluten is everywhere, and it’s use has increased enormously over the last few decades. It’s used as an ingredient in most processed food, to give texture - I am based in Italy, in a salumeria recently I was choosing some prosciutto, there were two types (actually being Italy there were probably about 20 types, just using licence here) and I asked the difference: one was free of phosphates while the other was also free of lactose and gluten – what the hell was gluten doing in ham in the first place, in Italy of all places?!

Some testing options:

Prometheus have a fairly expensive test at $329
Genelex at even more - $445
Decode and 23andme do not have SNPs that comprehensively type the HLA alleles but do have some other slight risk alleles discovered by GWAS. Not sure about Navigenics and Pathway as I couldn’t find the SNPs on their sites.

A promising recent development was published in PLOS One (Monsuur et al,  2009) where they describe 6 tagging SNPs that comprehensively genotype the relevant HLA alleles – any test based on these SNPs would be a lot cheaper than the PCR based methods used by Prometheus – e.g. 23andme could add them to their scan (as long as there are no exorbitant licence fees since the authors have applied for a patent on the findings … that’s another story for another day…)