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716
Immunodeficiency and Autoimmune Enterocolopathy Linked to NFAT5
Deficiency
Brigid S. Boland, Christella E. Widjaja, Asoka Banno, Stephanie H. Kim, Michael R.
Peterson, Marilyn C. Jones, H. I. Su, Sheila E. Crowe, Jack Bui, Jose F. Aramburu,
Cristina Lopez-Rodriguez, William Sandborn, John T. Chang
Recent genome-wide association studies have uncovered genetic links for a number of
complex diseases, but these powerful tools have not yet been widely applied to the clinical
management of disease. Here we describe our experience using a genetic approach to uncover
new molecular insights in a young patient presenting with unexplained infections and chronic
intestinal inflammation whom we eventually diagnosed with anti-goblet cell antibody positive
autoimmune enterocolopathy. Using a single-nucleotide polymorphism (SNP) array, we
identified a deletion of the gene Nuclear Factor of Activated T cells-5 (NFAT5) in one
chromosome in the patient and verified this deficiency at the mRNA and protein levels. In
mice, NFAT5 has previously been shown to regulate cellular responses to osmotic stress
and to play a critical role in influencing differentiation of pro-inflammatory T helper 17
(Th17) lymphocytes. We demonstrate for the first time that human NFAT5 deficiency
appears to result in functional defects in both innate and adaptive immunity, with a markedly
reduced number of natural killer (NK) cells, and impaired proliferation and cytokine produc-
tion by T lymphocytes. We confirm the molecular link between immune cell function and
NFAT5 deficiency using human cells as well as cells from mice deficient in NFAT5. Together
our approach and observations demonstrate the potential power of genetic analyses to
facilitate clinical diagnosis and to provide novel mechanistic insights into disease.
717
Loss of MTGR1 Accelerates Intestinal Tumor Burden
Bobak Parang, Amber Bradley, Mukul K. Mittal, Kay Washington, Frank Revetta, Anthony
Bilotta, J. Joshua Smith, Xi Chen, Noah F. Shroyer, Christopher S. Williams
Background: Myeloid translocation gene related-1 (MTGR1) is a transcriptional co-repressor
in the MTG/ETO family. MTGR1 complexes with DNA binding proteins and recruits other
coreperessors and histone deacetylases to modulate transcription of target genes. Mtgr1-/-
mice are deficient in intestinal secretory lineages. MTGR1 competes with TCF4 for β-catenin
occupancy to modulate TCF4 transcriptional activity. Thus, we hypothesized that MTGR1
deficiency might modify Wnt dependent intestinal tumorigenesis. Methods: A Vanderbilt
TMA consisting of 8 normal and 91 cancer samples was stained for MTGR1 and scored in
a blinded fashion. RNA expression was analyzed using Vanderbilt/Moffit CRC array consisting
of 10 normal, 6 adenomas, and 250 carcinomas. The mutant Apc1638 allele was introduced
into MTGR1 deficient mice. All experimental mice were Apc1638/+
. Mice were sacrificed at
36 weeks (Mtgr1+/+
N=36, Mtgr1-/+
N=25, and Mtgr1-/-
N=7), and tumors were counted
grossly and harvested for RNA. After sacrifice, intestines were stained for β-catenin and
phospho-histoneH3 (pH3). β-catenin was quantified using an index scoring nuclear localiza-
tion and intensity. Results: To determine whether MTGR1 expression is modified in colon
cancer (CRC), we stained a CRC TMA and found that MTGR1 was downregulated 2-fold
at the protein level (P<0.01). This relationship extended to its RNA levels as MTGR1 was
also downregulated in a large multistage Vanderbilt/Moffit colorectal cancer expression array
in adenomas and all stages of carcinomas (P<0.001). Perhaps most importantly, MTGR1
expression predicted patient survival (P<0.01). To evaluate the functional significance of
this observation Mtgr1-/-
and Apc1638/+
mice were crossed. MTGR1 loss increased mortality
and tumor multiplicity (WT 3.5± 0.4, Mtgr1+/-
6.8± 1.1, Mtgr1-/-
29± 4.5 intestinal tumors
per mouse, P<0.001) in an MTGR1 gene dose-dependent manner. Tumors from Mtgr1-/+
and Mtgr1-/-
mice were more dysplastic and progressed to invasive adenocarcinoma. Prolifera-
tion was determined by staining for pH3 and was increased in Mtgr1-/-
mice (Mtgr1-/-
140±
57 vs. WT 76± 36 positive cells/tumor P<0.001). Staining for intra-tumoral β-catenin, used
as a surrogate marker for Wnt activation, identified increases in both nuclear β-catenin and
intensity, indicating aberrant Wnt activity in the Mtgr1-/-
background (Mtgr1-/-
127± 17
vs. WT 79± 10 P<0.001). Intratumoral transcriptome analysis using RNA-seq supported
hyperactive Wnt signaling in Mtgr1-/-
tumors. Conclusions: MTGR1 is downregulated in
CRC and could be used as a predictor of patient survival. This is functionally relevant as
loss of MTGR1 increased tumor multiplicity and dysplasia possibly through deregulation of
Wnt signaling.
S-125 AGA Abstracts
Figure 1 Loss of MTGR1 Accelerates Intestinal Tumorigenesis A) Tumor multiplicity
of Apc1638/+
;Mtgr1+/+
, Apc1638/+
;Mtgr1+/-
,, and Apc1638/+
;Mtgr1-/-
, B) Histopathological analysis
of tumor dysplasia (Fisher's Exact Test ***P<0.001). c) Proliferation (phospho-histone H3)
D) Representative images of β-catenin immunohistochemistry and quantification of β-catenin
nuclear localization and intensity. *P<0.05,**P<0.01, ***P<0.001
Figure 2 MTGR1 is underexpressed in human CRC and predicts survival A) MTGR1
protein expression in Vanderbilt Tissue Microarray of CRC. B) Mtgr1 expression in Moffit/
Vanderbilt Ingram Cancer Center expression array and C) survival correlation in patients
with high or low mRNA. *P<0.05,**P<0.01, ***P<0.001
718
Whole Exome Sequencing in Cohort of Very Early Onset Inflammatory Bowel
Disease
Christopher J. Moran, Judith R. Kelsen, Jess L. Kaplan, Mariah Baril-Dore, Elizabeth Petit,
Tawfiq Al-Lawati, Batia Vais, Ramnik J. Xavier, Barbara S. Kirschner, Robert Baldassano,
Harland Winter, Mark J. Daly
BACKGROUND: Many of the genetic loci implicated in adult-onset inflammatory bowel
disease (IBD) have also been shown to play a role in pediatric-onset IBD. However, an
evolving literature of rare loss-of-function genetic variants in children with very early-onset
IBD (VEO-IBD) (such as mutations in interleukin-10 (IL-10), IL-10 receptor (IL-10R), and
x-linked inhibitor of apoptosis (XIAP)). We hypothesized that patients with VEO-IBD or
with extreme gastrointestinal presentations were likely to harbor rare, causative genetic
variants. METHODS: Patients with IBD with onset <6years old (and their parents) were
recruited along with other probands with extreme gastrointestinal symptoms. Following
informed consent salivary or whole blood DNA was collected. Exome capture was performed
by Agilent Whole Exome SureSelect kit and sequencing was performed on Illumina HiSeq.
Sequences were processed by Picard pipeline and aligned with GRCh37, h19 and variant
calling was performed using the GATK toolkit. Filters were applied to exclude variants with
a minor allele frequency >1% in the Exome Sequencing Project, 1000 Genomes Project,
and a control set from the Analytic and Translational Genetics Unit of Massachusetts General
Hospital. The functional significance of missense variants was estimated by SIFT and Pol-
yphen2 algorithms. Families were analyzed by either recessive (de novo, compound heterozy-
gous, and recessive homozygous) or dominant pattern based on known family history.
RESULTS: 70 probands with IBD were sequenced (including 30 with both parents available)
and included in the analysis. A novel, frameshift mutation in RIPK2 (chromosome 8,
90784949 T>TA) was identified in a patient with aggressive, ileocecal Crohn's disease (CD)
whose father possessed both that variant and a similarly aggressive CD phenotype. In
addition, a novel,homozygous, in-frame insertion variant in LXRB(chromosome 19,
50881822 A>ACAA) was identified in affected siblings. We also identified a homozygous,
in-frame deletion in SMPD1 (implicated in Niemann-Pick disease) in an infant with severe,
secretory diarrhea and hepatomegaly and as well as an infant with severe, infantile food
protein-induced enterocolitis as being a compound heterozygote for cma1. Functional analy-
sis in these families is on-going. CONCLUSION: Exome sequencing of patients with very
early onset IBD and other extreme gastrointestinal disease is likely to identify causative
variants although the therapeutic implications of these findings remain to be determined.
CJM and JRK contributed equally. HSW and MJD contributed equally.
719
Expression Quantitative Trait Loci Mapping in the Human Colon
Imge Hulur, Andrew Skol, Xavier Llor, Kenan Onel, Nathan A. Ellis, Sonia Kupfer
Background: Expression quantitative trait loci (eQTL) mapping is a tool to localize genomic
regions that regulate transcription and characterize variants identified through genome-wide
association studies (GWAS). While eQTL mapping studies have been performed in peripheral
blood, this tissue type may not be relevant for diseases primarily affecting the colon such
as colorectal cancer (CRC) and inflammatory bowel disease (IBD). Aim: To comprehensively
map eQTLs in the human colon in order to characterize colon-specific gene regulation and
to test for enrichment of GWAS variants from studies in CRC and IBD. Methods: 40 healthy
African American subjects undergoing colonoscopy were included. Colonic biopsies were
obtained at the rectosigmoid junction. Genotyping was performed with DNAs from blood
using Affymetrix Axiom Pan-African array. SNPs mapping to non-unique locations, SNP
with < 95% genotype call rates, minor allele frequency (MAF) < 5% or HWE p-value < 10-
6
were excluded. Additional genotypes were determined by imputation using 1000 Genomes
data with IMPUTE2. After imputation, SNPs with info scores < 0.5 and MAF < 5% were
further excluded. Gene expression was profiled using Illumina HT-12v4 Expression BeadChip
whole-genome expression array. After background correction and normalization of expression
array data, eQTL mapping was performed using the Matrix eQTL package. Associations
between gene expression and SNP genotypes were analyzed using a linear regression model
controlling for ancestry and the first 4 principal components of the expression data. Significant
cis eQTLs (FDR < 0.1) were identified and compared to SNPs associated with either CRC
or IBD in GWAS. Results: After quality control, 8.4 million autosomal SNPs and 16,252
expression probes were included in the analysis. We noted strong enrichment for cis eQTLs
in the colon (Fig 1). At an FDR<0.1, there were 8,382 significant cis eQTLs-gene pairs of
which 1,171 were independent. We noted enrichment for cis eQTLs near the transcription
start site. In total, there were 6 colonic disease-associated SNPs that were significant eQTLs
in the colon (Fig 2). The two IBD-associated SNPs rs4266763 and rs10640, common to
AGAAbstracts

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PIIS0016508514604509

  • 1. 716 Immunodeficiency and Autoimmune Enterocolopathy Linked to NFAT5 Deficiency Brigid S. Boland, Christella E. Widjaja, Asoka Banno, Stephanie H. Kim, Michael R. Peterson, Marilyn C. Jones, H. I. Su, Sheila E. Crowe, Jack Bui, Jose F. Aramburu, Cristina Lopez-Rodriguez, William Sandborn, John T. Chang Recent genome-wide association studies have uncovered genetic links for a number of complex diseases, but these powerful tools have not yet been widely applied to the clinical management of disease. Here we describe our experience using a genetic approach to uncover new molecular insights in a young patient presenting with unexplained infections and chronic intestinal inflammation whom we eventually diagnosed with anti-goblet cell antibody positive autoimmune enterocolopathy. Using a single-nucleotide polymorphism (SNP) array, we identified a deletion of the gene Nuclear Factor of Activated T cells-5 (NFAT5) in one chromosome in the patient and verified this deficiency at the mRNA and protein levels. In mice, NFAT5 has previously been shown to regulate cellular responses to osmotic stress and to play a critical role in influencing differentiation of pro-inflammatory T helper 17 (Th17) lymphocytes. We demonstrate for the first time that human NFAT5 deficiency appears to result in functional defects in both innate and adaptive immunity, with a markedly reduced number of natural killer (NK) cells, and impaired proliferation and cytokine produc- tion by T lymphocytes. We confirm the molecular link between immune cell function and NFAT5 deficiency using human cells as well as cells from mice deficient in NFAT5. Together our approach and observations demonstrate the potential power of genetic analyses to facilitate clinical diagnosis and to provide novel mechanistic insights into disease. 717 Loss of MTGR1 Accelerates Intestinal Tumor Burden Bobak Parang, Amber Bradley, Mukul K. Mittal, Kay Washington, Frank Revetta, Anthony Bilotta, J. Joshua Smith, Xi Chen, Noah F. Shroyer, Christopher S. Williams Background: Myeloid translocation gene related-1 (MTGR1) is a transcriptional co-repressor in the MTG/ETO family. MTGR1 complexes with DNA binding proteins and recruits other coreperessors and histone deacetylases to modulate transcription of target genes. Mtgr1-/- mice are deficient in intestinal secretory lineages. MTGR1 competes with TCF4 for β-catenin occupancy to modulate TCF4 transcriptional activity. Thus, we hypothesized that MTGR1 deficiency might modify Wnt dependent intestinal tumorigenesis. Methods: A Vanderbilt TMA consisting of 8 normal and 91 cancer samples was stained for MTGR1 and scored in a blinded fashion. RNA expression was analyzed using Vanderbilt/Moffit CRC array consisting of 10 normal, 6 adenomas, and 250 carcinomas. The mutant Apc1638 allele was introduced into MTGR1 deficient mice. All experimental mice were Apc1638/+ . Mice were sacrificed at 36 weeks (Mtgr1+/+ N=36, Mtgr1-/+ N=25, and Mtgr1-/- N=7), and tumors were counted grossly and harvested for RNA. After sacrifice, intestines were stained for β-catenin and phospho-histoneH3 (pH3). β-catenin was quantified using an index scoring nuclear localiza- tion and intensity. Results: To determine whether MTGR1 expression is modified in colon cancer (CRC), we stained a CRC TMA and found that MTGR1 was downregulated 2-fold at the protein level (P<0.01). This relationship extended to its RNA levels as MTGR1 was also downregulated in a large multistage Vanderbilt/Moffit colorectal cancer expression array in adenomas and all stages of carcinomas (P<0.001). Perhaps most importantly, MTGR1 expression predicted patient survival (P<0.01). To evaluate the functional significance of this observation Mtgr1-/- and Apc1638/+ mice were crossed. MTGR1 loss increased mortality and tumor multiplicity (WT 3.5± 0.4, Mtgr1+/- 6.8± 1.1, Mtgr1-/- 29± 4.5 intestinal tumors per mouse, P<0.001) in an MTGR1 gene dose-dependent manner. Tumors from Mtgr1-/+ and Mtgr1-/- mice were more dysplastic and progressed to invasive adenocarcinoma. Prolifera- tion was determined by staining for pH3 and was increased in Mtgr1-/- mice (Mtgr1-/- 140± 57 vs. WT 76± 36 positive cells/tumor P<0.001). Staining for intra-tumoral β-catenin, used as a surrogate marker for Wnt activation, identified increases in both nuclear β-catenin and intensity, indicating aberrant Wnt activity in the Mtgr1-/- background (Mtgr1-/- 127± 17 vs. WT 79± 10 P<0.001). Intratumoral transcriptome analysis using RNA-seq supported hyperactive Wnt signaling in Mtgr1-/- tumors. Conclusions: MTGR1 is downregulated in CRC and could be used as a predictor of patient survival. This is functionally relevant as loss of MTGR1 increased tumor multiplicity and dysplasia possibly through deregulation of Wnt signaling. S-125 AGA Abstracts Figure 1 Loss of MTGR1 Accelerates Intestinal Tumorigenesis A) Tumor multiplicity of Apc1638/+ ;Mtgr1+/+ , Apc1638/+ ;Mtgr1+/- ,, and Apc1638/+ ;Mtgr1-/- , B) Histopathological analysis of tumor dysplasia (Fisher's Exact Test ***P<0.001). c) Proliferation (phospho-histone H3) D) Representative images of β-catenin immunohistochemistry and quantification of β-catenin nuclear localization and intensity. *P<0.05,**P<0.01, ***P<0.001 Figure 2 MTGR1 is underexpressed in human CRC and predicts survival A) MTGR1 protein expression in Vanderbilt Tissue Microarray of CRC. B) Mtgr1 expression in Moffit/ Vanderbilt Ingram Cancer Center expression array and C) survival correlation in patients with high or low mRNA. *P<0.05,**P<0.01, ***P<0.001 718 Whole Exome Sequencing in Cohort of Very Early Onset Inflammatory Bowel Disease Christopher J. Moran, Judith R. Kelsen, Jess L. Kaplan, Mariah Baril-Dore, Elizabeth Petit, Tawfiq Al-Lawati, Batia Vais, Ramnik J. Xavier, Barbara S. Kirschner, Robert Baldassano, Harland Winter, Mark J. Daly BACKGROUND: Many of the genetic loci implicated in adult-onset inflammatory bowel disease (IBD) have also been shown to play a role in pediatric-onset IBD. However, an evolving literature of rare loss-of-function genetic variants in children with very early-onset IBD (VEO-IBD) (such as mutations in interleukin-10 (IL-10), IL-10 receptor (IL-10R), and x-linked inhibitor of apoptosis (XIAP)). We hypothesized that patients with VEO-IBD or with extreme gastrointestinal presentations were likely to harbor rare, causative genetic variants. METHODS: Patients with IBD with onset <6years old (and their parents) were recruited along with other probands with extreme gastrointestinal symptoms. Following informed consent salivary or whole blood DNA was collected. Exome capture was performed by Agilent Whole Exome SureSelect kit and sequencing was performed on Illumina HiSeq. Sequences were processed by Picard pipeline and aligned with GRCh37, h19 and variant calling was performed using the GATK toolkit. Filters were applied to exclude variants with a minor allele frequency >1% in the Exome Sequencing Project, 1000 Genomes Project, and a control set from the Analytic and Translational Genetics Unit of Massachusetts General Hospital. The functional significance of missense variants was estimated by SIFT and Pol- yphen2 algorithms. Families were analyzed by either recessive (de novo, compound heterozy- gous, and recessive homozygous) or dominant pattern based on known family history. RESULTS: 70 probands with IBD were sequenced (including 30 with both parents available) and included in the analysis. A novel, frameshift mutation in RIPK2 (chromosome 8, 90784949 T>TA) was identified in a patient with aggressive, ileocecal Crohn's disease (CD) whose father possessed both that variant and a similarly aggressive CD phenotype. In addition, a novel,homozygous, in-frame insertion variant in LXRB(chromosome 19, 50881822 A>ACAA) was identified in affected siblings. We also identified a homozygous, in-frame deletion in SMPD1 (implicated in Niemann-Pick disease) in an infant with severe, secretory diarrhea and hepatomegaly and as well as an infant with severe, infantile food protein-induced enterocolitis as being a compound heterozygote for cma1. Functional analy- sis in these families is on-going. CONCLUSION: Exome sequencing of patients with very early onset IBD and other extreme gastrointestinal disease is likely to identify causative variants although the therapeutic implications of these findings remain to be determined. CJM and JRK contributed equally. HSW and MJD contributed equally. 719 Expression Quantitative Trait Loci Mapping in the Human Colon Imge Hulur, Andrew Skol, Xavier Llor, Kenan Onel, Nathan A. Ellis, Sonia Kupfer Background: Expression quantitative trait loci (eQTL) mapping is a tool to localize genomic regions that regulate transcription and characterize variants identified through genome-wide association studies (GWAS). While eQTL mapping studies have been performed in peripheral blood, this tissue type may not be relevant for diseases primarily affecting the colon such as colorectal cancer (CRC) and inflammatory bowel disease (IBD). Aim: To comprehensively map eQTLs in the human colon in order to characterize colon-specific gene regulation and to test for enrichment of GWAS variants from studies in CRC and IBD. Methods: 40 healthy African American subjects undergoing colonoscopy were included. Colonic biopsies were obtained at the rectosigmoid junction. Genotyping was performed with DNAs from blood using Affymetrix Axiom Pan-African array. SNPs mapping to non-unique locations, SNP with < 95% genotype call rates, minor allele frequency (MAF) < 5% or HWE p-value < 10- 6 were excluded. Additional genotypes were determined by imputation using 1000 Genomes data with IMPUTE2. After imputation, SNPs with info scores < 0.5 and MAF < 5% were further excluded. Gene expression was profiled using Illumina HT-12v4 Expression BeadChip whole-genome expression array. After background correction and normalization of expression array data, eQTL mapping was performed using the Matrix eQTL package. Associations between gene expression and SNP genotypes were analyzed using a linear regression model controlling for ancestry and the first 4 principal components of the expression data. Significant cis eQTLs (FDR < 0.1) were identified and compared to SNPs associated with either CRC or IBD in GWAS. Results: After quality control, 8.4 million autosomal SNPs and 16,252 expression probes were included in the analysis. We noted strong enrichment for cis eQTLs in the colon (Fig 1). At an FDR<0.1, there were 8,382 significant cis eQTLs-gene pairs of which 1,171 were independent. We noted enrichment for cis eQTLs near the transcription start site. In total, there were 6 colonic disease-associated SNPs that were significant eQTLs in the colon (Fig 2). The two IBD-associated SNPs rs4266763 and rs10640, common to AGAAbstracts