In our previous work, we considered in CD families the risk to develop the disease according to a specific HLA haplotypes, obtaining a risk range from 0.01 to ≥0.20 
. In the present study we evaluated the role of 3 non-HLA genetic markers to influence the CD risk in first relatives of CD affected children.
We collected data on families with at least one CD-affected among offspring. This family set helped to evaluate the association between SNPs and CD (TDT on parents-offspring trios) and to estimate the risk of CD in the other sibs. The TDT design provides robustness to population stratification and mitigation of the possible confounding effect of environmental factors, because all family members share the same environment 
Ten SNPs, selected from those previously found to be associated with CD by GWAS 
, were successfully genotyped. In our population three SNPs resulted significantly associated with CD (those in LPP, RGS1 and REL genes) and the other seven investigated SNPs, even if not statistically associated with CD, showed always an higher frequency of the previously reported risk alleles 
in affected subjects than in controls.
The three genes selected appear to be appealing for the pathogenesis of CD. LPP (OR
2.36; p<0.001) was reported to be highly expressed in small intestinal mucosa and may have a structural role at sites of cell adhesion in maintaining cell shape and motility 
. RGS1 (OR
0.025) belongs to a family of RGS genes. It attenuates the signaling activity of G-proteins, blocking the homing of Intra Epithelial Lymphocytes (IELs), and it is specifically expressed both in human small intestinal mucosa and in murine IELs, key players in the development of human CD villous atrophy 
. REL (OR
0.034) is a subunit of NF-kB complex, implicated in T cell differentiation 
and it appears to be a key molecule regulating inflammation and the switch from tolerance to autoimmunity 
. It is interesting to note that our data confirm previous pathogenetic implications reported in literature of these SNPs with CD as well as with other autoimmune diseases 
By the Bayesian approach we calculated a ranking score (BS) among the sibs. However, it should be considered that BS is not a plain disease risk, rather a method to rank different genotypes according to their contribution to make an individual susceptible to CD. For instance, some of our BS are very near to 1, nevertheless none of the considered genotypes could give a 100% risk to develop the disease. In other terms, we considered the BS as a ranking measure, only stating that a given genotype could assign a higher risk than another genotype but does not allow a quantitative measure of the risk difference (2-fold, 3-fold, etc). However, even if the addition of only 3 SNPs to HLA could be considered at “minor effect” 
, we demonstrated that they could significantly improve the prediction of CD risk in sibs, in terms of diagnostic sensitivity and negative predictive value. So, in a cohort of CD families, our data confirm that non-HLA SNPs evaluation is an usefull diagnostic tool in CD risk evaluation as a previous study showed in CD unrelated subjects 
CD, on the basis of the actual knowledge, cannot be exactly predicted by genetic testing, but a reliable probabilistic method might be associated to careful surveillance of infants carrying the higher risk. This will help to significantly reduce the heavy load of anxiety and pain associated with the appearance of symptoms of CD, by anticipating, with simple serological tests, the clinical appearance of the disease.
To improve the possibility to identify high risk patients in CD families we propose in alternative to the classical HLA classification (, panel A) a slight improved flow-chart (, panel B): 1) HLA genotyping: subjects belonging to the HLA risk groups 1 and 2 will be classified as at high CD risk; 2) subjects belonging to the HLA risk groups 3 and 4, will be further investigated for our SNPs combination (LPP, REL, RGS1) in order to calculate their BS (, panel B). Among these latter subjects those with a BS ≥ the median value will be classified at high risk; 3) subjects belonging to the HLA risk group 5 will be considered at low CD risk. All CD familials belonging to the above high risk groups (HLA group 1–2 and HLA group 3–4 with BS ≥ median) will be undergo a strict surveillance.
One of the limitation of our cohort family study could be the sample size, which may have not allowed to explore genes at smaller effect, so explaining the lack of association between SNPs in TAGAP, IL2/IL21, OLIG3, CCR, SH2B3, IL12A and IL12A/SCHIP1 genes with CD although the trend observed in previous studies in unrelated CD patients was confirmed 
. In the main time the homogeneity of the genetic and environmental domains in the tested families allows to explore risk factors within a controlled cohort. A second limit of the study is the relatively short (6 years) follow up of the sibship, which could cause an underestimation of the disease development at later ages. Our aim is to go on with the monitoring of these families in the next years.
In conclusion, the estimate of the CD risk by HLA+SNPs approach, even if not applicable to prevention, could be a precious tool improving CD diagnosis respect to the only HLA (NPV: 95% vs 91%, and DS: 79% vs 45%), in the cohort of first degree relatives. In fact in clinical practice the absence of HLA risk groups 1 or 2, allows to exclude the disease with high probability, while testing the three SNPs in HLA groups 3 or 4 could represent a further tool to identify less frequent CD cases. So, an infant with high HLA+SNPs score even if belonging to HLA low risk groups, shall undergo a simple surveillance system to allow proper diagnosis and treatment before the full blow disease appears.