Novel genetic risk variants for pediatric celiac disease
- Angeliki Balasopoulou†1,
- Biljana Stanković†2,
- Angeliki Panagiotara1,
- Gordana Nikčevic2,
- Brock A. Peters3, 4,
- Anne John5,
- Effrosyni Mendrinou1,
- Apostolos Stratopoulos1,
- Aigli Ioanna Legaki1,
- Vasiliki Stathakopoulou1,
- Aristoniki Tsolia1,
- Nikolaos Govaris1,
- Sofia Govari1,
- Zoi Zagoriti1,
- Konstantinos Poulas1,
- Maria Kanariou6,
- Nikki Constantinidou6,
- Maro Krini7,
- Kleopatra Spanou6,
- Nedeljko Radlovic8,
- Bassam R. Ali5,
- Joseph Borg9,
- Radoje Drmanac3, 4,
- George Chrousos7,
- Sonja Pavlovic2,
- Eleftheria Roma7,
- Branka Zukic2,
- George P. Patrinos1, 5 and
- Theodora Katsila1Email author
© The Author(s). 2016
Received: 10 August 2016
Accepted: 16 October 2016
Published: 24 October 2016
Celiac disease is a complex chronic immune-mediated disorder of the small intestine. Today, the pathobiology of the disease is unclear, perplexing differential diagnosis, patient stratification, and decision-making in the clinic.
Herein, we adopted a next-generation sequencing approach in a celiac disease trio of Greek descent to identify all genomic variants with the potential of celiac disease predisposition.
Analysis revealed six genomic variants of prime interest: SLC9A4 c.1919G>A, KIAA1109 c.2933T>C and c.4268_4269delCCinsTA, HoxB6 c.668C>A, HoxD12 c.418G>A, and NCK2 c.745_746delAAinsG, from which NCK2 c.745_746delAAinsG is novel. Data validation in pediatric celiac disease patients of Greek (n = 109) and Serbian (n = 73) descent and their healthy counterparts (n = 111 and n = 32, respectively) indicated that HoxD12 c.418G>A is more prevalent in celiac disease patients in the Serbian population (P < 0.01), while NCK2 c.745_746delAAinsG is less prevalent in celiac disease patients rather than healthy individuals of Greek descent (P = 0.03). SLC9A4 c.1919G>A and KIAA1109 c.2933T>C and c.4268_4269delCCinsTA were more abundant in patients; nevertheless, they failed to show statistical significance.
The next-generation sequencing-based family genomics approach described herein may serve as a paradigm towards the identification of novel functional variants with the aim of understanding complex disease pathobiology.
Celiac disease is a complex chronic immune-mediated disorder of the small intestine. Today, the pathobiology of the disease is unclear, perplexing differential diagnosis, patient stratification, and decision-making in the clinic. Genetics has been reported to play a key role. The HLA-DQ2 gene is identified in up to 95 % of celiac disease patients, while most of the remaining patients have the HLA-DQ8 gene. Notwithstanding, the Chinese and Japanese populations (devoid of HLA-DQ2) are not expected to develop the disease, yet this is not true for the individuals with the HLA-DQ8 gene. Celiac disease is also associated with an extended ancestral haplotype that is defined by class I and II HLAs (A, B, DR, DQ). Notably, HLA-DQ2 and/or HLA-DQ8 expression is necessary but not sufficient for disease development. Thus, other genes are anticipated to be involved. Indeed, genome-wide association studies (GWAS) have revealed 26 non-HLA genetic loci-associated celiac disease and other autoimmune or chronic immune disorders (diabetes mellitus type I, rheumatoid arthritis) [1, 2]. In 2008 to 2011, several new celiac disease risk loci have been identified [3–5], bringing the number of known loci (including the HLA one) to 40 and indicating genes and gene regulatory elements of paramount importance. In 2015, five new genetic loci were identified, being independent of HLA-DQA1 and HLA-DQB1 and associated with celiac disease predisposition .
Although a genetic component has been described, disease occurrence has been also associated with environmental factors and gut microbiome. In all cases, gluten has been identified as the environmental trigger of the disease, leading to the stimulation of gluten-specific T cells. Differential diagnosis is still a major issue. Although a gold standard diagnostic approach has been defined (endoscopy with biopsy of the small intestine coupled to positive disease serology), several pathological conditions have been reported sharing similar mucosal transformations with celiac disease as well as other autoimmune disorders (thyroid disease, Addison disease, autoimmune liver disease, Sjögren syndrome) that occur ten times more frequently in celiac disease patients often masking celiac disease symptoms. Disease management options are restricted to a gluten-free lifestyle, which ultimately fails to protect patients from disease symptoms due to its chronic nature. Can we delineate individual variability towards differential diagnosis? Can we highlight the disease mechanisms in question to assist disease management?
So far, findings account for 49 % of the genetic basis of the disease. As in other immune-mediated diseases, genetic predisposition to celiac disease remains unresolved as we still need to explain the remaining major fraction of heritability, including rare as well as additional common risk variants. Causal variants and genes still need to be identified and/or more finely localized. In this context, the Immunochip Consortium was developed to explore comprehensive datasets containing common, low-frequency, and rare variants in related diseases (autoimmune thyroid disease, ankylosing spondylitis, Crohn disease, celiac disease, IgA deficiency, multiple sclerosis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes mellitus, and ulcerative colitis) .
As expected, the advent of technology and, in particular, next-generation sequencing has provided unprecedented opportunities to delineate disease pathobiology as well as inter-individual differences [8, 9]. Herein, we propose a multi-step next-generation sequencing-based family genomics approach, piloted in a celiac disease trio of Greek descent to identify novel genomic variants of functional significance with the aim of understanding disease pathobiology.
Case selection, DNA isolation, and whole-genome sequencing
A seven-member Greek family has been recruited (informed consents have been obtained), and a trio analysis (III-1, III-2, IV-3) has been performed using the celiac disease model (Additional file 1: Figure S1). A family-based design was employed rather than a population-based design, as the former is generally considered to be robust against population admixture and stratification and may yield both within- and between-family information . Genomic DNA isolation was performed from saliva using the Oragene collection kit (DNA Genotek, Ontario, Canada) (Serbian cohort) and peripheral blood using an automated system (MagNA Pure Compact, Roche, Basel, Switzerland) (Greek cohort). Whole-genome sequencing was performed using Complete Genomics’ (CA, USA) DNA nanoarray platform . DNA sequencing coverage was 110×. Only high-quality call variants were included in the analysis (>93 %). Genomes were aligned with the hg19 reference genome.
Bioinformatics and in silico analyses
Next-generation sequencing data (Complete Genomics Inc., CA) were analyzed using Ingenuity Variant Analysis version 3.1.2 (Ingenuity® Systems, www.ingenuity.com). This is a well-established software that identifies associations between phenotypes, defined by the user by classification of the tested individuals, and variants in the sequenced genome. Upon classification of the family members by phenotype (celiac vs. normal), a number of variants were listed; the output was filtered into a smaller variant list upon classification of the family members by those being celiac patients vs. those who were healthy and known not to be celiac disease subjects. The genetic model used for this comparison was of an autosomal dominant model, since it traces the genetic inheritance from mother (III-2) to daughter (IV-3) in a highly penetrant form. A total of 263 genes followed an autosomal dominant pattern, and 227 variants were identified in the genetic model. Out of these, 6 genes and 7 variants were identified in the biological/phenotype pattern of celiac disease, due to either the past association of the genes considered or via the biological significance as determined by the IVA software.
Confidence: Only variants with call quality at least 20.0 in cases or at least 20.0 in controls (i.e., 99 % call accuracy) were included.
Common variants: All variants observed to have an allele frequency ≥3.0 % of the genomes in the 1000 genomes project OR ≥3.0 % of the public Complete Genomics genomes OR ≥3.0 % of the NHLBI ESP exomes were excluded.
Predicted deleterious: Only variants that are experimentally observed to be associated with a phenotype (Pathogenic, Possibly Pathogenic, Unknown Significance OR established gain of function in the literature OR gene fusions OR inferred activating mutations by Ingenuity OR predicted gain of function by BSIFT OR in a microRNA binding site OR Frameshift, in-frame indel, or stop codon change OR Missense and not predicted to be innocuous by SIFT OR disrupt splice site up to 2.0 bases into intron OR deleterious to a microRNA OR structural variant) were kept.
Genetic analysis: Only variants with the following genotype characteristics were kept: associated with gain of function OR heterozygous_alt OR haploinsufficient OR heterozygous OR homozygous OR heterozygous_amb OR compound_heterozygous OR hemizygous AND occur in at least 2 of the case samples at the gene level in the Case samples AND not which are associated with gain of function OR heterozygous_alt OR haploinsufficient OR heterozygous OR homozygous OR heterozygous_amb OR compound_heterozygous OR hemizygous AND occur in at least 1 of the control samples at the variant level in the control samples.
Biological context: Only variants that are within 1 hop (direct targets) of upstream regulators or downstream regulatory targets of such genes and that are known or predicted to affect celiac disease.
Variants of interest were annotated with Annovar in Galaxy  and compared with NCBI dbSNP build 137 (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_summary.cgi), 69 reference genomes from Complete Genomes (http://www.completegenomics.com/publicdata/69Genomes/), and GWAS (http://www.genome.gov/gwastudies) to determine their novelty or otherwise. To obtain a list of variants of potential functional significance, we employed protein variation effect analyzer (PROVEAN) v1.1.3 (PROVEAN human genome variants tool) that provides both scale-invariant feature transform (SIFT)  and PROVEAN  predictions for a given list of human genome variants as well as accessory information (dbSNP rs IDs, gene description, PFAM domain, GO terms, etc.). PROVEAN is able to make predictions for any type of protein sequence alteration, including single or multiple amino acid substitutions, deletions, and insertions . Additionally, Variant Effect Predictor  and RegulomeDB  were employed to allow further data interrogation.
Downstream molecular analysis
Selected variants were subsequently validated in pediatric celiac disease patients of Greek (n = 109)  and Serbian (n = 73)  descent and their healthy counterparts (n = 111 and n = 32, respectively). The diagnosis of celiac disease was based on the criteria of the European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) . For children diagnosed prior to 1990, the “Interlaken criteria” were applied. The Ethics Committee of University Children’s Hospital, University of Belgrade, and the Review Board of “Aghia Sophia” Children’s Hospital have approved the study.
Amplification was carried out according to the KAPA2G Fast HotStart protocol (KAPABIOSYSTEMS, MA, USA); detailed information per SNP amplification conditions is available upon request. For SLC9A4 c.1919G>A, an allele-specific polymerase chain reaction (PCR) assay was developed (two alternative reverse primers hybridizing exclusively either to the wild-type or the mutant allele). For NCK2 c.745_746delAAinsG, PCR products were subjected to XcmI (New England Biolabs, MA, USA) restriction endonuclease analysis at 37 °C for 1.15 h and subsequent enzyme deactivation (65 °C, 20 min). Restriction fragments were visualized by 3 % agarose gel electrophoresis following ethidium bromide or Midori Green staining. For HoxD12 c.418G>A, HoxB6 c.668C>A, and KIAA1109 c.2933T>C and c.4268_4269delCCinsTA, a PCR-based conventional Sanger resequencing approach was employed. Capillary electrophoresis was performed on the ABI Prism 3130xl DNA Analyzer (Applied Biosystems). Sanger sequencing was also employed to ensure PCR-RFLP and allele-specific PCR method verification.
Herein, a thorough statistical review and analysis has been attempted, having always in mind that we used the celiac disease model (trio analysis) as a reason to conduct a more refined approach in searching/genotyping the resulted variants in a selective population of celiac disease of Greek and Serbian descent. We tested for deviations from HWE using the chi-square goodness of fit test and principal component analysis. Considering that the χ 2 approximation can be poor when there are low genotype counts, a Fisher exact test (R genetics package) was also used, as it does not rely on the χ 2 approximation . Tests were performed as two-tailed, and differences were considered statistically significant when P < 0.05. Focusing on case-control phenotypes, we tested the null hypothesis of no association between rows and columns of the 2 × 3 matrix that contains the counts of the three genotypes (the two homozygotes and the heterozygote) among cases and controls. Again, a Fisher exact test was preferred (to evaluate genotype and allele frequencies), as it is computationally more demanding, but it is easily implemented in R. We also performed the Armitage test (Monte Carlo method; it obtains results that are closer to an exact test, since the classical Cochran-Armitage trend test is based on approximation) .
Results and discussion
Whole-genome sequencing analysis of trio reveals newly identified genetic risk variants for pediatric celiac disease
In silico analyses
In silico analyses outcome of the six variants of prime interest identified in the family trio
HUGO gene symbol
HGVS description of variant
Variant effect predictor
Single AA change
Single AA change
Single AA change
Single AA change
Single AA change
Single AA change
Genotyping data of celiac disease pediatric patients of Greek and Serbian descent and healthy individuals
Genotype frequency (%)
Healthy individuals of Greek descent
Patients of Greek descent
Healthy individuals of Serbian descent
Patients of Serbian descent
In relation to disease diagnosis and prognosis, data interpretation requires an understanding of the variation in risk-associated variants. In celiac disease, in particular, this knowledge is still largely lacking. As whole-genome and/or whole-exome sequencing approaches begin to be employed in clinical care, the understanding of detected sequence variations on diagnosis (and prognosis) is still not a trivial undertaking. We envisage that the clinical implementation of next-generation sequencing will play a crucial role in delineating inter-individual variability and identification of novel variants towards improved therapeutic modalities. Herein, we propose a multi-step next-generation sequencing-based family genomics approach, similar to our previous conducted cancer genomics study , but piloted towards a complex genetic disease, such as celiac disease, to analyze a family trio of Greek descent to identify novel genomic variants of functional significance with the aim of understanding complex disease pathobiology. Recently, we have outlined the paradigm of pharmacometabolomics-aided pharmacogenomics in autoimmune diseases to address the interplay of genomic and environmental influences with information technologies to facilitate data analysis as well as sense- and decision-making on the basis of synergy between artificial and human intelligence. We propose that better-informed, rapid, and cost-effective “omics” studies need the implementation of holistic and multidisciplinary approaches .
This study was funded by the European Commission (RD-CONNECT; FP7-304555) research grant to GPP.
Availability of data and materials
The datasets during and/or analyzed during the current study are available from the corresponding author on request.
TK and GPP conceived and designed the study. AB, AP, BS, GN, AJ, EM, AS, AIL, VS, AT, NG, SG, ZZ, MK, MK, KS, and TK carried out the PCR-based conventional Sanger resequencing approach. BAP and RD supervised the next-generation sequencing analyses. TK carried out the in silico analyses. BRA, NC, ER, JB, KP, GC, BZ, NR, RD, SP, TK, and GPP drafted the manuscript. All authors read and approved the final manuscript.
The authors declare the following competing interests: BAP and RD are employees of the Complete Genomics Inc. (Mountain View, CA, USA).
Consent for publication
Ethics approval and consent to participate
A family trio of Greek descent was recruited for this study (informed consents have been obtained). The Ethics Committee of University Children’s Hospital, University of Belgrade, and the Review Board of “Aghia Sophia” Children’s Hospital have approved the study.
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