Molecular diagnosis of infantile onset inflammatory bowel disease by exome sequencing

Abstract

Pediatric-onset inflammatory bowel disease (IBD) is known to be associated with severe disease, poor response to therapy, and increased morbidity and mortality. We conducted exome sequencing of two brothers from a non-consanguineous relationship who presented before the age of one with severe infantile-onset IBD, failure to thrive, skin rash, and perirectal abscesses refractory to medical management. We examined the variants discovered in all known IBD-associated and primary immunodeficiency genes in both siblings. The siblings were identified to harbor compound heterozygous mutations in IL10RA (c.784C>T, p.Arg262Cys; c.349C>T, p.Arg117Cys). Upon molecular diagnosis, the proband underwent successful hematopoietic stem cell transplantation and demonstrated marked clinical improvement of all IBD-associated clinical symptoms. Exome sequencing can be an effective tool to aid in the molecular diagnosis of pediatric-onset IBD. We provide additional evidence of the safety and benefit of HSCT for patients with IBD due to mutations in the IL10RA gene.

1. Introduction

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the intestinal tract composed of two somewhat clinically distinct disorders, Crohn's disease and ulcerative colitis. Worldwide it is estimated to affect approximately 2.5 million people. IBD is a result of an inappropriate and uncontrolled inflammatory response to commensal intestinal microbes [1]. Despite a well-recognized genetic component, IBD is a complex multifactorial disorder characterized by a genetic predisposition, but in general, development of disease is still dependent upon the interactions of these genetic variations with the gut and intestinal flora, and environmental factors [2].

To date, genome wide association studies (GWAS) have identified at least 163 loci encompassing more than 300 genes that are associated with IBD [3]. Genes in at least seven distinct, but interrelated, pathways have been found to be associated with IBD through GWAS studies, including those involved in the epithelial barrier and junctions, innate immune sensors, g-protein coupled receptors, IL-10 signaling, the Th17 pathway, T-cell negative regulators, and B-cell function [1,2,4]. Within these pathways multiple genes involved in cellular responses including autophagy, ER stress, intracellular logistics, cell migration, apoptosis/necroptosis, carbohydrate metabolism, and oxidative stress have been implicated. Furthermore, genes more directly involved in immune-mediated intestinal homeostasis including the epithelial barrier, restitution, solute transport, Paneth cells, innate mucosal defense, immune cell recruitment, antigen presentation, IL-13/Th17, T-cell regulation, B-cell regulation, and immune tolerance also play a critical function in IBD [1,2,4]. Despite the success of GWAS at discovering pathways involved in the pathophysiology of IBD, the studies have largely failed to identify actual causal variants.

However, multiple monogenic causes of IBD with causal variants of high effect have been recently identified. Mutations in the gene encoding the X-linked inhibitor of apoptosis (XIAP) [5] have linked to early-onset severe colitis and the interleukin 10 (IL10) [6] and its associated receptor alpha and beta subunits (IL10RA and IL10RB) [7] have been shown to cause very-early-onset IBD (VEO-IBD). The Paris classification defines early-onset IBD (EO-IBD) as diagnosis before the 18th birthday, VEO-IBD as diagnosis before the age of six years old, and infantile-onset IBD as diagnosis before the 1st birthday [8]. It is likely that genetic factors contribute more to the etiology of IBD in cases where multiple family members are affected, in infantile-onset and VEO-IBD, and in cases demonstrating extreme clinical phenotypes. To that end, we enrolled two siblings with onset of IBD prior to one year of age, which was refractory to medical management into the Children's Mercy Undiagnosed Disease Research Program to undergo exome sequencing.

2. Case presentations

The proband, CMH000166, is an 8 year old male who presented at 6 months of age with perirectal abscess and failure to thrive. Within the first year of life he developed anemia, diarrhea, failure to thrive and skin rash (Table 1, Supplement Fig. 1). Endoscopic evaluation revealed features consistent with Crohn's disease affecting his upper GI tract and colon. There were multiple perianal cutaneous abscesses and colon biopsy revealed crypt destruction, crypt distortion and occasional crypt abscessed with patchy basal plasmacytosis (Fig. 1). Additionally, skin biopsy revealed neutrophilic dermatosis. Medical treatment with azathioprine, infliximab (10 mg/kg), adalimumab and, finally, tacrolimus failed to adequately control his disease, resulting in corticosteroid dependence. Enteral nutrition was attempted but failed to restore normal growth velocity. Refractory, severe disease resulted in a colectomy at age 3, ileal resection at age 5, and over 20 hospitalizations. CMH000165 is the 16 month old younger brother of the proband who presented at two months of age with oral lesions and decreased oral food intake, and was seen again at four months of age with oral lesions, anemia, failure to thrive and perianal skin rash (Table 1, Supplement Fig. 2). Biopsies obtained at Esophagogastroduodenoscopy and flexible sigmoidoscopy revealed esophagitis and colitis with the presence of granulomas. He is corticosteroid dependent, having failed tacrolimus and infliximab. Currently he is treated with 1 mg/kg of prednisone, elemental diet, night time parenteral nutrition, azathioprine and adalimumab, yet is still affected with persistent inflammation and perianal disease and has a weight z-score of —4. Both siblings had a significant medical history of increased susceptibility to infections consistent with a primary immunodeficiency (PID). CMH000166 had three separate methicillin-sensitive Staphylococcus aureus (MSSA) infections and multiple upper respiratory and sinus infections by the age of 5 years. The younger sibling, CMH000165, had three infections of Enterococcus faecalis and multiple severe rotavirus, rhinovirus, parainfluenza virus and respiratory syncytial virus infections. Immunological evaluation of CMH000166 revealed mildly elevated IgA at 262 mg/dl² (normal 13.9–105 mg/dl²), but normal IgG, IgM, C3 and C4 levels. In the younger sibling, CMH000165, slightly elevated IgG, IgM, IgA and IgE levels were noted.

Table 1.

Clinical summary of patients.

	CMH000166	CMH000165
Age	8 years	16 months
Gender	Male	Male
Onset of symptoms	6 months	2 months
Perianal disease	+	+
Extent of disease	Upper disease proximal and distal to the ligament of trietz; ileocolonic	Upper disease proximal to the ligament of trietz; colonic
Behavior of disease	Stricturing and penetrating	Stricturing and penetrating
Extraintestinal manifestations	Neutrophilic dermatosis, osteoporosis	Neutrophilic dermatosis

Fig. 1.

Clinical findings of CMH000166. Perianal abscesses (A, B), colon biopsy with crypt destruction (C), crypt abscess and basal plasmacytosis (D).

3. Results

Given the severe and early onset of disease in a sibling pair, a monogenic cause of either autosomal recessive or X-linked inheritance was suspected. Consequently, both parents and the siblings were enrolled in a research protocol to have their exomes sequenced by the Center for Pediatric Genomic Medicine (CPGM) at Children's Mercy Hospital [9-11]. Briefly, DNA was isolated from peripheral blood of the affected siblings and their non-consanguineous, nonaffected parents and enriched for 62.2 million bases representing the coding regions of the genome (Illumina v1) and sequenced by Illumina next-generation sequencing. Alignment, variant detection, and characterization were performed as previously described [10]. The exomes of CMH000165 and CMH000166 received 10.7 and 11.9 gigabases of sequence which resulted in mean target coverages of 83.4× and 92.2×, respectively (Supplement Table 1). Less than 3% of the targeted exome received 0× coverage, and 90% received 20× coverage (Supplement Table 1). A total of 116,020 variants were detected in CMH000165 and 117,583 variants were detected in CMH000166 (Supplement Table 2). Variant characterization by RUNES resulted in 24 category 1 variants in CMH000165 and 27 category 1 variants in CMH000166 (Supplement Table 2). Analysis of category 1 variants revealed that both siblings were compound heterozygous for two missense mutations in the IL10RA gene. Mutation 1 was inherited from the mother (c.784C>T, p.Arg262Cys, Supplement Fig. 3) [12] and mutation 2 was inherited from the father (c.349C>T, p.Arg117Cys, Supplement Fig. 4) [13]. The siblings were found to share 11 category 2 variants with an allele frequency of less than 1%. However, all of the identified category 2 variants were heterozygous and not consistent with an autosomal recessive or X-linked inheritance pattern. Furthermore, none of the category 2 variants were in genes associated with disease and thus, were all deemed likely to be nonpathogenic and unrelated to the clinical phenotype (Supplement Table 3).

To further exclude other potential high effect IBD-associated rare variants we examined the variants discovered in both siblings in 301 genes (Supplement Table 4) associated with IBD through GWAS studies [3]. In addition, we wanted to investigate potential causes of the immune-related dysfunction seen in our siblings that might be due to mutations other than the IL10RA mutations discovered; therefore, we analyzed variants found in 90 known primary immunodeficiency (PID) genes (Supplement Table 5) [14]. Given the familial inheritance pattern, the severity of IBD in our patients, and the known low frequency of PIDs in the population we decided to examine only rare variants in coding or splice sites with an allele frequency of less than 1%. Consequently, only nonsynonymous, insertion/deletion, and variants that were predicted to impact splicing and that were found to be at a frequency of 1% or less in dbSNP v137 [15], the 1000 Genomes Project [16] or CPGM internal variant database [9,10], were evaluated. Using these filtering criteria 338 variants in CMH000165 and 377 variants in CMH000166 were analyzed. Surprisingly, only four variants were found in the IBD-associated genes (Supplement Table 6) and zero variants were discovered in either sibling in the 90 PID genes analyzed. Of the four variants discovered in IBD-associated genes, three were shared by the siblings. Further analysis of the four genes revealed only two, TXK tyrosine kinase and chemokine (C–C motif) receptor 6, with demonstrated function in pathways known to be associated with IBD; however, both variants were previously seen in patients sequenced at CMH who did not have clinical symptoms of IBD and consequently were judged to be nondisease-causing. Taken together, the presence of two mutations with functional characterization and previously reported in VEO-IBD patients with the lack of additional disease-causing mutations in 301 genes associated with IBD by GWAS and 90 known PID genes the two mutations in IL10RA were deemed sufficient to be diagnostic. Consequently, confirmatory clinical capillary sequencing was conducted and the molecular diagnostic results were returned to the treating physicians.

There is compelling, but limited data, suggesting the clinical utility of hematopoietic stem cell transplantation (HSCT) to induce remission of clinical symptoms in patients with IL10R mutations [7,13,17]. At the time of molecular diagnosis, CMH000166 had a weight Z-score of −3.91, his height plateau Z-score was −2.48, and his bone density T-score was −3.1. The patient was on tacrolimus and 2 mg/kg of prednisone. A magnetic resonance enterography (MRE) revealed narrowing of the ileum which was going to require bowel resection. In response to CMH000166's severe clinical condition and previous reports of treatment by HSCT, his parents decided to proceed with the procedure. An unrelated 10 out of 10 match was found. Briefly, the reparatory regimen for transplant consisted of Campath, busulfan and fludarabine and graft versus host prophylaxis consisted of tacrolimus and mycophenolate (Fig. 2). He was given antibacterial prophylaxis with metronidazole and ciprofloxacin during and after transplantation. The patient engrafted on day 21 post-HSCT as indicated by short tandem repeat analysis of peripheral blood (98% donor cells). He required one platelet transfusion, with an adverse reaction of rash, hypotension, and angioedema with swelling of the throat and tongue. His immediate post-transplant course was complicated by severe rotavirus diarrhea and Enterococcus hirae and Klebsiella pneumoniae bacteremia, necessitating hospital readmission on day 24 post-HSCT. Following intensive treatment to control the infections, the patient was released from the hospital on day 71 post-transplant. Further complications occurred on day 100 post-transplant when the patient presented with a low grade fever and swelling on the right side of the neck due to painful lymphadenopathy. Epstein bar virus (EBV) PCR showed 2500 copies in the plasma. A repeat PCR three days later showed 17,000 copies of EBV. Ultrasound of the abdomen revealed enlargement of the spleen and porta-hepatis lymph node enlargement. A computed tomography (CT) scan of the chest revealed three nodular opacities with the largest being 4 mm in the left upper lobe. Biopsy of lymph nodes from the neck confirmed monomorphic clonal B cell post-transplant lymphoproliferative disorder (PTLD). The patient was treated with two doses of Rituxan weekly and withdrawal of immunosuppressants. His lymphadenopathy subsequently resolved, and EBV viremia subsided and he was released from the hospital.

Fig. 2.

Hematopoietic stem cell transplant preparatory regiment.

Subsequently, to better understand the rare, nonsynonymous variant burden and impact of mutations in the IL10RA gene, we examined the number of variants of a frequency of less than 1% in the IL10RA gene in the CPGM internal variant database [9,10]. Briefly, this database keeps track of every variant detected at the CPGM, at what frequency the said variant has been seen and the clinical phenotype of each patient that the variant was found in. In more than 1300 exomes sequenced at the CPGM, 19 nonsynonymous variants with a frequency of less than 1% have been identified (Supplement Table 7). Of these, 42% are predicted to be deleterious by SIFT (Sorts Intolerant From Tolerant substitutions) [18] and 37% as possibly damaging by PolyPhen2 (Polymorphism Phenotyping 2) [19] (Supplement Table 7). Interestingly, in the CPGM database the only samples that contain two predicted deleterious and possibly damaging variants are the siblings reported here and none of the patients harboring a single, heterozygous damaging variant exhibited clinical IBD symptoms.

4. Discussion

Definitive molecular diagnosis of VEO-IBD is especially challenging due to locus heterogeneity and overlapping signs and symptoms of monogenic and complex forms of IBD. However, increasing evidence suggests that rare, highly penetrant genetic variants may exist for many common diseases [20-22]. Whole exome and genome analysis of familial cases is one method to discover these. Currently, diagnosis of affected individuals with VEO-IBD and other rare genetic disorders is lengthy and costly – the “diagnostic odyssey” – and in many patients a definitive molecular diagnosis is never achieved despite extensive clinical investigation. In response to these needs, our hospital started an undiagnosed childhood disease program two and a half years ago, and has enrolled over 500 individuals. They received genome, exome, or targeted panel sequencing and analysis at no cost in addition to extensive conventional clinical investigation.

Here, we report the clinical utility of exome sequencing to determine the molecular cause of infantile-onset IBD in siblings, which enabled personalized therapy. Both mutations found in our patients have previously been reported independently, p.Arg117Cys in a compound heterozygous patient [13] and p.Arg262Cys as a homozygous mutation in a patient with VEO-IBD [12]; however, this is the first report of these two mutations together causing VEO-IBD. Importantly, in contrast to most reports to date [6,7,13,23], our siblings are from a non-consanguineous union and highlight that a monogenic cause of VEO-IBD should not be overlooked when the parents are not related. Of note, the duration from the time of enrollment in the undiagnosed disease program to the time of return of molecular results to the physician was approximately one month demonstrating the potential speed of diagnosis through exome sequencing. In addition, we present further evidence that IL10 receptor mutations should be considered in the differential diagnosis of infantile-onset and VEO-IBD, particularly in patients with severe phenotypes including aggressive disease, perianal lesions and dermatosis.

By conducting whole exome sequencing we were able to make a more definite diagnosis than a targeted approach might provide because we were able to exclude more potential mutations and exclude other causes of the clinical symptoms. We found only four variants with a frequency of less than 1% in 301 IBD-associated genes (Supplement Table 6). Of the four variants discovered in IBD-associated genes, three were shared by the siblings; however, zero variants were judged to be disease-causing in their heterozygous state as they were previously seen in patients sequenced at CMH who did not have clinical symptoms of IBD. Furthermore, we were also able to exclude a secondary cause of immune dysfunction by analyzing 90 primary immunodeficiency genes. Our analysis did not reveal a single known or likely disease-causing mutation in any of these genes in either sibling, providing further evidence to the support the mutations in the IL10RA as the likely cause of the immune dysfunction. However, it should be cautioned that it can be difficult to distinguish primary immune dysfunction as a result of impaired IL10 signaling from those related to the immunosuppressive treatments that most of the children with severe IBD are on.

We provide further evidence of the benefit of HSCT for patients with VEO-IBD due to mutations in the IL10RA gene. CMH000166 underwent a successful HSCT that leads to the clinical remission of his IBD; however, it was not without complications. Within days of release from the hospital a severe rotavirus infection required readmission to the hospital. Interestingly, sequencing of the rotavirus infection revealed a wild-type strain possibly contracted from his younger sibling. Previous studies have also reported severe viral infections in IL10R patients post-transplantation [7,13,17], suggesting that this patient population may be at risk for atypical post-transplant viral infections. In addition, CHM000166 also developed post-transplant lymphoproliferative disorder. Despite the complications of the HSCT marked clinical improvement in the child was noted, including increased weight gain, improvement in energy and endurance, and complete elimination of GI symptoms and dermatitis seven months post-transplant. A stem cell donor has been identified for the younger sibling, CMH000165, and a HSCT is being scheduled.

A recent study found IL10R polymorphisms to be associated with very-early-onset ulcerative colitis [24]. In the CPGM's internal exome database with more than 1300 samples we found 11 variants in the IL10RA including the two reported here and only 2 variants in the IL10RB gene with a allele frequency of less than 1%, suggesting that damaging mutations in these two genes are indeed infrequent. Of note, none of the samples with mutations in either gene had IBD-associated clinical symptoms mentioned and two were found in healthy parents of children with an unrelated rare disease.

Definitive molecular diagnosis has several potential benefits that we've previously discussed [25]. First, in this family it provided a treatment option that might not otherwise have been considered. HSCT is arduous and not without risk, but at the time of molecular diagnosis two separate reports [7,13] suggested in patients with IL10 pathway mutations that it was an effective and potentially the only long-term treatment option. With this information, both the family and physicians were able to weigh the risk-benefits and make an informed clinical management decision. Second, identifying a molecular cause of a monogenic disorder allows for genetic counseling about recurrence risks in the family and their relatives. In this case, the family now knows that any additional pregnancy has a 25% chance of the child also being affected. Third, it helps improve variant databases with regard to both pathogenicity of specific variants and frequency within a population. This, the second and third reports of patients with VEO-IBD harboring these specific mutations add evidence to their pathogenicity.

In summary, the clinical and genetic heterogeneity of VEO-IBD makes it a particularly good candidate for exome sequencing; however there is insufficient data to date on the clinical yield in VEO-IBD. We report at least in an isolated familial case with infantile-onset IBD that exome sequencing is indeed beneficial for molecular diagnostic and clinical management purposes. However, additional studies are needed to examine the specificity and sensitivity of genome or exome sequencing before they are considered a primary diagnostic tool for patients with VEO-IBD.

5. Methods

5.1. Consent

The study was approved by the Institutional Review Board of Children's Mercy Hospital (CMH). Informed written consent was obtained from adult subjects and assent was obtained from the parents of the children.

5.2. Targeted exome sequencing

Exome sequencing was performed at the Center for Pediatric Genomic Medicine (CPGM) at CMH under a research protocol. Isolated genomic DNA was prepared for sequencing using the Kapa Biosystems library preparation kit and 8 cycles of PCR amplification. Exome enrichment was conducted with the Illumina TruSeq Exome v1 kit (62.2 megabases) following a slightly modified version of the manufacturer recommended protocol. The enrichment protocol was modified to use the Kapa Biosystems PCR amplification kit for the post-enrichment amplification step to limit polymerase induced GC-bias [26]. Successful enrichment was verified by qPCR of 4 targeted loci and 2 non-targeted loci of the sequencing library pre- and post-enrichment prior to sequencing [27]. The enriched library was sequenced on an Illumina HiSeq 2000 using v3 reagents and 1 × 101 base pair sequencing reads.

5.3. Next generation sequencing analysis

Sequence data was generated with Illumina RTA 1.12.4.2 & CASAVA-1.8.2, aligned to the human reference NCBI 37 using the Genomic Short-read Nucleotide Alignment Program (GSNAP) [28] and variants were detected and genotyped using the Genome Analysis Toolkit (GATK) [29]. Sequence analysis employed FASTQ files, the compressed binary version of the Sequence Alignment/Map format (bam, a representation of nucleotide sequence alignments) and Variant Call Format (VCF, a format for nucleotide variants). Variants were characterized with the CPGM's Rapid Understanding of Nucleotide variant Effect Software (RUNES v1.0) [10]. RUNES incorporates data from the Variant Effect Predictor (VEP) software [30], and produces comparisons to NCBI dbSNP, known disease mutations from the Human Gene Mutation Database [31] and performs additional in silico prediction of variant consequences using ENSEMBL and UCSC gene annotations [32,33]. RUNES categorizes each variant according to the American College of Medical Genetics (ACMG's) recommendations for reporting sequence variation [34,35] as well as an allele frequency derived from CPGM's Variant Warehouse database [10]. Briefly, category 1 variants are those previously described as disease-causing, category 2 variants are those of the type likely to disrupt protein function and be disease-causing if they are in a gene associated with disease, and category 3 variants are those of unknown significance that may or may not cause disease.

5.4. Capillary sequencing

Primers and PCR conditions are available upon request. PCR products were purified using Exo-Sapit (USB Corporation, Cleveland, OH) according to the manufacturer's instructions. Both the forward and reverse strands of the purified PCR product were sequenced using fluorescent dye-terminator sequencing. Sequencing reactions were purified using the BigDye XTerminator Purification Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Results were analyzed on an ABI 3130 analyzer (Applied Biosystems, Foster City, CA). Sequence results were compared to published reference sequence (NM_001558.3) using Sequencher 4.5 (Gene Codes Corporation, Ann Arbor, MI).

Abbreviations

ACMG

American College of Medical Genetics

CMH

Children's Mercy Hospital

CPGM

Center for Pediatric Genomic Medicine

CT

Computed tomography

EBV

Epstein bar virus

EO-IBD

Early-onset inflammatory bowel disease

GATK

Genome analysis toolkit

GI

Gastrointestinal

GSNP

Genomic short-read nucleotide alignment program

GWAS

Genome wide association studies

HSCT

Hematopoietic stem cell transplantation

IBD

Inflammatory bowel disease

IL10

Interleukin 10

IL10RA

Interleukin 10 receptor, alpha

IL10RB

Interleukin 10 receptor, beta

PCR

Polymerase chain reaction

PID

Primary immunodeficiency

PTLD

Post-transplant lymphoproliferative disease

qPCR

Quantitative polymerase chain reaction

SIFT

Sorts intolerant from tolerant substitutions

VEO-IBD

Very-early-onset inflammatory bowel disease

XIAP

X-linked inhibitor of apoptosis