EDITORIALS Exome Sequencing in Familial Colorectal Cancer: Searching for Needles in Haystacks See “Germline mutation of RPS20, encoding a ribosomal protein, causes predisposition to hereditary nonpolyposis colorectal carcinoma without DNA mismatch repair deficiency,” by Nieminen TT, O’Donohue M-F, Wu Y, et al, on page 595.
The way to find the needle in the haystack is to sit down. – Beryl Markham, West with the Night
N
early one third of all colorectal cancers (CRCs) are diagnosed in individuals who have a family history of the disease.1 A family history that fulfills the Amsterdam criteria (CRCs diagnosed in 3 individuals, spanning 2 generations, with 1 diagnosed at age 6000 population-based controls. These segregation data and in vitro functional assays suggest this alteration results in a defect in pre-rRNA maturation and is the likely cause of cancer in this family. It has been < 25 years since use of linkage analysis and positional cloning resulted in discovery of highly penetrant genetic mutations in families with multiple individuals affected with CRC in a pattern suggestive of autosomal dominant inheritance. The vast majority of these Mendelian hereditary CRC syndromes are associated with germline mutations in either DNA mismatch repair genes or tumor suppressor genes and to date there are more than a dozen genes known to be associated with clinically significant increases in risk for CRC, most of which exhibit autosomal dominant patterns of inheritance (Table 1). However, germline mutations are estimated to play a role in only 5%–6% of CRC cases, which Gastroenterology 2014;147:554–565
suggests 2 possible explanations for familial CRC. First, there are additional as-of-yet undiscovered, highly penetrant genes responsible for cancer risk. Second, cancer risk in familial CRC is the result of less penetrant genetic alterations, heritable nongenetic factors, environmental factors, or a combination of these. The variability in expressivity of disease among carriers of the same germline mutations in cancer predisposition genes (within and between families) has prompted investigations into additional genetic and/or environmental factors which may act as modifiers of cancer risk. Next-generation sequencing (NGS) technologies make it possible to accomplish, relatively quickly and inexpensively, genetic investigations which previously took years. Massively parallel exome sequencing (WES) analyzes the entire coding regions of >20,000 individual genes and has facilitated discovery of germline mutations implicated in risk for various tumor types, such as MAX in pheochromocytoma6 and PALB2 in familial pancreatic cancer.7 However, until now no novel genes have emerged as the “smoking gun” for FCCX. In their search for a genetic cause of FCCX, Nieminen et al employed basic Mendelian genetics and NGS technologies to analyze DNA from multiple individuals from a single family. It is worth noting that WES identified >1,000 variants in each individual, and filtering of these variants was facilitated greatly by comparing the data from multiple affected family members, because only 2 variants were shared among 4 CRC-affected relatives, and only 1 segregated perfectly with disease status. Had the variant in RPS20 been one of many variants shared among siblings, or had it been seen in only one of many unrelated probands with FCCX, the investigators may not even have considered it a potential candidate because, until now, mutations in RPS genes have been associated primarily with cases of Diamond–Blackfan anemia.8 However, the fact that the truncating mutation in one allele of RPS20 was the only variant shared among all CRC-affected individuals provides compelling evidence of its potential association with autosomal dominantly inherited risk for CRC in this family. Questions remain, however, about how RPS20 influences cancer risk. What is the mechanism by which genes which encode for ribosomal proteins might predispose to cancer? If RSP20 is truly causal in FCCX, why were variants in this gene (or any of the other 80 ribosomal genes) not identified in any of the other 12 FCCX families studied? How important is this gene in the broader picture of FCCX? Despite these unanswered questions, this study illustrates the tremendous potential of exome sequencing for discovery and highlights the continued importance of family studies in genetics. When Palles et al employed whole genome sequencing in probands from 13 APC mutationnegative attenuated polyposis families, initial analyses failed to identify any variants shared among 4 families;
EDITORIALS Table 1.Genes Associated With Inherited Predisposition to Colorectal Cancer (CRC) Gene(s)
Mechanism
Syndrome
Pattern of inheritance
MLH1 MSH2 MSH6 PMS2 EPCAM/TACSTD1
DNA mismatch repair (MMR)
Lynch syndrome
AD
APC
Tumor suppressor
Familial adenomatous polyposis (FAP)
AD (with de-novo mutations implicated in 30% of cases)
MutYH
Base-excision repair
MutYH-associated polyposis (MAP)
AR
SMAD4 BMPR1A
Tumor suppressor
Juvenile polyposis
AD
STK11/LKB1
Tumor suppressor
Peutz–Jeghers syndrome
AD
PTEN
Tumor suppressor
PTEN hamartoma tumor syndrome (PHTS)/Cowden syndrome
AD
P53
Tumor suppressor
Li Fraumeni syndrome
AD
CDH1
Tumor suppressor
Hereditary diffuse gastric cancer (HDGC)
AD
POLE POLD1
DNA replication and repair
To be defined
AD
AD, autosomal dominant; AR, autosomal recessive.
however, when investigators analyzed results among several individuals from the same family, they identified common variants in POLE and POLD1.9 Validation of associations between mutations in these genes and CRC risk in larger cohorts has led to their inclusion in some NGS multigene panels currently available for clinical genetic testing. Although the potential role for RPS20 in FCCX was similarly identified through sequencing of siblings, the fact that this mutation has been implicated in FCCX in only a single family is insufficient to prove causality, and further validation is needed before RPS20 is added to the list of cancer predisposition genes for which clinical genetic testing is available. Nevertheless, it is worth pointing out that these approaches to identifying candidate genes through examination of single families represent a (successful) departure from strategies applied in most genome-wide association studies (GWAS) investigating genetic factors associated with CRC risk. The search for candidate genes in GWAS is based on the assumption that the causal variants are not rare, but rather that they are more commonly found among affected cases compared with unaffected controls. Although GWAS conducted in thousands of individuals have identified dozens of single nucleotide polymorphisms associated with an increased risk for CRC, these universally have been associated with small effect sizes (odds ratios < 1.5).10 Given that most genotyping arrays used for GWAS include single nucleotide polymorphisms with allele frequencies of >5% in 1000 genomes, rare variants in genes such as RPS20 that do not appear in 1000 genomes would not be expected to ever make it onto the stage. Considering that the exome comprises >20,000 genes representing only 1%–2% of the
whole human genome, WGS approaches can produce a haystack of variants that is 100 times larger still. If we assume that there is 1 gene in this haystack that can be implicated in the vast majority of familial CRC (the common variant common disease hypothesis11), then we might expect approaches which employ GWAS or exome sequencing of probands from different families to readily identify this needle. However, these approaches have produced limited insight into the pathogenesis of FCCX, which should prompt reconsideration of the experimental design. Perhaps the variant filtering algorithms currently in use are not sophisticated enough to differentiate needle(s) from shiny straw. Perhaps we lack a clear idea of what kind of needle we are searching for. Or perhaps our search should not be focused on identifying just 1 single needle. Illustratively, exome/genome sequencing misses all heritable nongenetic factors such as germline methylation.12 Like familial pancreatic cancer, which seems to lack a unifying genetic basis, FCCX cases are similarly defined primarily on the basis of family history rather than phenotype and it remains possible that these may be multifactorial in etiology. Instead of assuming a model of common disease–common variant, we might consider the alternative hypotheses that FCCX is caused by multiple, rare variants or that it is not a single disease, but actually multiple different diseases with different causal factors. Consequently, instead of searching for a single needle in a single haystack, we might actually need to expand our search of genetic causes of FCCX to consider the possibility that there may actually be many different needles, and that each family may constitute its own haystack. Although the Amsterdam Criteria suggest 555
EDITORIALS autosomal-dominant inheritance, whether FCCX is a monogenic or a polygenic disease remains unclear and the role of gene–environment interactions and epigenetic modifications remain largely unexplored. The diagnostic criteria for FCCX (family history and microsatellite stable CRC tumors) are nonspecific and have significant potential overlap with not only sporadic CRCs clustering by chance, but also other known Mendelian syndromes. Interestingly, mutations in BMPR1A (associated with juvenile polyposis syndrome) were identified in 2 of 18 FCCX families (11%) in Nieminen et al’s original cohort.13 The increasing use of multigene panels in the clinical setting will help to define how many FCCX cases may be attributable to mutations in genes already known to be associated with cancer predisposition. The experience of Nieminen et al highlights the “agony and ecstasy” of modern genetics research. NGS technologies generate massive amounts of genetic data and bioinformatics and computational strategies must continue to evolve to enable us to analyze these complex data effectively. Family studies continue to be valuable for providing a framework on which to overlay genetic data. The search for a needle in a haystack is difficult even when you have an idea of the category of gene you are looking for; however, when confronted with 1000 gene variants in each subject, any one of which might be “real,” the value of being able to compare data from affected and unaffected family members to inform variant filtering and annotation cannot be underestimated. A major question which remains is how many common highly penetrant variants are yet to be discovered. As information regarding germline and somatic variants in different diseases continues to increase, it is possible we will discover novel pathways involved in neoplasia not previously on our radar for CRC or malignancy. FCCX is most likely etiologically heterogeneous. It is almost certain that the RPS20 and CRC risk connection is private to a single family or shared by a small subset of FCCX families. At the fundamental level, this is an exciting observation because it reveals an entire new class of genes under candidacy for other types of cancer risk. ELENA M. STOFFEL Division of Gastroenterology Department of Internal Medicine University of Michigan Health System Ann Arbor, Michigan CHARIS ENG Genomic Medicine Institute and Taussig Cancer Institute Cleveland Clinic and Department of Genetics and Genome Sciences and Comprehensive Cancer Center Case Western Reserve University Cleveland, Ohio
References 1. Jasperson KW, Tuohy TM, Neklason DW, et al. Hereditary and familial colon cancer. Gastroenterology 2010; 138:2044–2058.
556
2. Vasen HF, Watson P, Mecklin JP, et al. New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC. Gastroenterology 1999; 116:1453–1456. 3. Boland CR, Goel A. Microsatellite instability in colorectal cancer. Gastroenterology 2010;138:2073–2087.e3. 4. Lindor NM, Rabe K, Petersen GM, et al. Lower cancer incidence in Amsterdam-I criteria families without mismatch repair deficiency: familial colorectal cancer type X. JAMA 2005;293:1979–1985. 5. Nieminen TT, O’Donohue M-F, Wu Y, et al. Germline mutation of RPS20, encoding a ribosomal protein, causes predisposition to hereditary nonpolyposis colorectal carcinoma without DNA mismatch repair deficiency. Gastroenterology 2014;147:595–598. 6. Comino-Mendez I, Gracia-Aznarez FJ, Schiavi F, et al. Exome sequencing identifies MAX mutations as a cause of hereditary pheochromocytoma. Nat Genet 2011;43: 663–667. 7. Jones S, Hruban RH, Kamiyama M, et al. Exomic sequencing identifies PALB2 as a pancreatic cancer susceptibility gene. Science 2009;324:217. 8. Mirabello L, Macari ER, Jessop L, et al. Wholeexome sequencing and functional studies identify RPS29 as a novel gene mutated in multi-case Diamond-Blackfan anemia families. Blood 2014; 124:24–32. 9. Palles C, Cazier JB, Howarth KM, et al. Germline mutations affecting the proofreading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas. Nat Genet 2013;45:136–144. 10. Tenesa A, Dunlop MG. New insights into the aetiology of colorectal cancer from genome-wide association studies. Nat Rev Genet 2009;10:353–358. 11. Manolio TA, Collins FS, Cox NJ, et al. Finding the missing heritability of complex diseases. Nature 2009;461: 747–753. 12. Bennett KL, Mester J, Eng C. Germline epigenetic regulation of KILLIN in Cowden and Cowden-like syndrome. JAMA 2010;304:2724–2731. 13. Nieminen TT, Abdel-Rahman WM, Ristimaki A, et al. BMPR1A mutations in hereditary nonpolyposis colorectal cancer without mismatch repair deficiency. Gastroenterology 2011;141:e23–26.
Reprint requests Address requests for reprints to: Elena M. Stoffel, MD, MPH, University of Michigan Health System, Division of Gastroenterology, 3912 Taubman Center, 1500 E. Medical Center Drive, Ann Arbor, Michigan 48109. e-mail: [email protected]. Conflicts of interest The authors disclose the following: Charis Eng is a member of the External Strategic Advisory Board of N-of-One and an unpaid member of the External Scientific Advisory Boards of Ecoeos and GenomOncology. Dr Stoffel discloses no conflicts. © 2014 by the AGA Institute 0016-5085/$36.00 http://dx.doi.org/10.1053/j.gastro.2014.07.031
The way to find the needle in the haystack is to sit down. – Beryl Markham, West with the Night
N
early one third of all colorectal cancers (CRCs) are diagnosed in individuals who have a family history of the disease.1 A family history that fulfills the Amsterdam criteria (CRCs diagnosed in 3 individuals, spanning 2 generations, with 1 diagnosed at age 6000 population-based controls. These segregation data and in vitro functional assays suggest this alteration results in a defect in pre-rRNA maturation and is the likely cause of cancer in this family. It has been < 25 years since use of linkage analysis and positional cloning resulted in discovery of highly penetrant genetic mutations in families with multiple individuals affected with CRC in a pattern suggestive of autosomal dominant inheritance. The vast majority of these Mendelian hereditary CRC syndromes are associated with germline mutations in either DNA mismatch repair genes or tumor suppressor genes and to date there are more than a dozen genes known to be associated with clinically significant increases in risk for CRC, most of which exhibit autosomal dominant patterns of inheritance (Table 1). However, germline mutations are estimated to play a role in only 5%–6% of CRC cases, which Gastroenterology 2014;147:554–565
suggests 2 possible explanations for familial CRC. First, there are additional as-of-yet undiscovered, highly penetrant genes responsible for cancer risk. Second, cancer risk in familial CRC is the result of less penetrant genetic alterations, heritable nongenetic factors, environmental factors, or a combination of these. The variability in expressivity of disease among carriers of the same germline mutations in cancer predisposition genes (within and between families) has prompted investigations into additional genetic and/or environmental factors which may act as modifiers of cancer risk. Next-generation sequencing (NGS) technologies make it possible to accomplish, relatively quickly and inexpensively, genetic investigations which previously took years. Massively parallel exome sequencing (WES) analyzes the entire coding regions of >20,000 individual genes and has facilitated discovery of germline mutations implicated in risk for various tumor types, such as MAX in pheochromocytoma6 and PALB2 in familial pancreatic cancer.7 However, until now no novel genes have emerged as the “smoking gun” for FCCX. In their search for a genetic cause of FCCX, Nieminen et al employed basic Mendelian genetics and NGS technologies to analyze DNA from multiple individuals from a single family. It is worth noting that WES identified >1,000 variants in each individual, and filtering of these variants was facilitated greatly by comparing the data from multiple affected family members, because only 2 variants were shared among 4 CRC-affected relatives, and only 1 segregated perfectly with disease status. Had the variant in RPS20 been one of many variants shared among siblings, or had it been seen in only one of many unrelated probands with FCCX, the investigators may not even have considered it a potential candidate because, until now, mutations in RPS genes have been associated primarily with cases of Diamond–Blackfan anemia.8 However, the fact that the truncating mutation in one allele of RPS20 was the only variant shared among all CRC-affected individuals provides compelling evidence of its potential association with autosomal dominantly inherited risk for CRC in this family. Questions remain, however, about how RPS20 influences cancer risk. What is the mechanism by which genes which encode for ribosomal proteins might predispose to cancer? If RSP20 is truly causal in FCCX, why were variants in this gene (or any of the other 80 ribosomal genes) not identified in any of the other 12 FCCX families studied? How important is this gene in the broader picture of FCCX? Despite these unanswered questions, this study illustrates the tremendous potential of exome sequencing for discovery and highlights the continued importance of family studies in genetics. When Palles et al employed whole genome sequencing in probands from 13 APC mutationnegative attenuated polyposis families, initial analyses failed to identify any variants shared among 4 families;
EDITORIALS Table 1.Genes Associated With Inherited Predisposition to Colorectal Cancer (CRC) Gene(s)
Mechanism
Syndrome
Pattern of inheritance
MLH1 MSH2 MSH6 PMS2 EPCAM/TACSTD1
DNA mismatch repair (MMR)
Lynch syndrome
AD
APC
Tumor suppressor
Familial adenomatous polyposis (FAP)
AD (with de-novo mutations implicated in 30% of cases)
MutYH
Base-excision repair
MutYH-associated polyposis (MAP)
AR
SMAD4 BMPR1A
Tumor suppressor
Juvenile polyposis
AD
STK11/LKB1
Tumor suppressor
Peutz–Jeghers syndrome
AD
PTEN
Tumor suppressor
PTEN hamartoma tumor syndrome (PHTS)/Cowden syndrome
AD
P53
Tumor suppressor
Li Fraumeni syndrome
AD
CDH1
Tumor suppressor
Hereditary diffuse gastric cancer (HDGC)
AD
POLE POLD1
DNA replication and repair
To be defined
AD
AD, autosomal dominant; AR, autosomal recessive.
however, when investigators analyzed results among several individuals from the same family, they identified common variants in POLE and POLD1.9 Validation of associations between mutations in these genes and CRC risk in larger cohorts has led to their inclusion in some NGS multigene panels currently available for clinical genetic testing. Although the potential role for RPS20 in FCCX was similarly identified through sequencing of siblings, the fact that this mutation has been implicated in FCCX in only a single family is insufficient to prove causality, and further validation is needed before RPS20 is added to the list of cancer predisposition genes for which clinical genetic testing is available. Nevertheless, it is worth pointing out that these approaches to identifying candidate genes through examination of single families represent a (successful) departure from strategies applied in most genome-wide association studies (GWAS) investigating genetic factors associated with CRC risk. The search for candidate genes in GWAS is based on the assumption that the causal variants are not rare, but rather that they are more commonly found among affected cases compared with unaffected controls. Although GWAS conducted in thousands of individuals have identified dozens of single nucleotide polymorphisms associated with an increased risk for CRC, these universally have been associated with small effect sizes (odds ratios < 1.5).10 Given that most genotyping arrays used for GWAS include single nucleotide polymorphisms with allele frequencies of >5% in 1000 genomes, rare variants in genes such as RPS20 that do not appear in 1000 genomes would not be expected to ever make it onto the stage. Considering that the exome comprises >20,000 genes representing only 1%–2% of the
whole human genome, WGS approaches can produce a haystack of variants that is 100 times larger still. If we assume that there is 1 gene in this haystack that can be implicated in the vast majority of familial CRC (the common variant common disease hypothesis11), then we might expect approaches which employ GWAS or exome sequencing of probands from different families to readily identify this needle. However, these approaches have produced limited insight into the pathogenesis of FCCX, which should prompt reconsideration of the experimental design. Perhaps the variant filtering algorithms currently in use are not sophisticated enough to differentiate needle(s) from shiny straw. Perhaps we lack a clear idea of what kind of needle we are searching for. Or perhaps our search should not be focused on identifying just 1 single needle. Illustratively, exome/genome sequencing misses all heritable nongenetic factors such as germline methylation.12 Like familial pancreatic cancer, which seems to lack a unifying genetic basis, FCCX cases are similarly defined primarily on the basis of family history rather than phenotype and it remains possible that these may be multifactorial in etiology. Instead of assuming a model of common disease–common variant, we might consider the alternative hypotheses that FCCX is caused by multiple, rare variants or that it is not a single disease, but actually multiple different diseases with different causal factors. Consequently, instead of searching for a single needle in a single haystack, we might actually need to expand our search of genetic causes of FCCX to consider the possibility that there may actually be many different needles, and that each family may constitute its own haystack. Although the Amsterdam Criteria suggest 555
EDITORIALS autosomal-dominant inheritance, whether FCCX is a monogenic or a polygenic disease remains unclear and the role of gene–environment interactions and epigenetic modifications remain largely unexplored. The diagnostic criteria for FCCX (family history and microsatellite stable CRC tumors) are nonspecific and have significant potential overlap with not only sporadic CRCs clustering by chance, but also other known Mendelian syndromes. Interestingly, mutations in BMPR1A (associated with juvenile polyposis syndrome) were identified in 2 of 18 FCCX families (11%) in Nieminen et al’s original cohort.13 The increasing use of multigene panels in the clinical setting will help to define how many FCCX cases may be attributable to mutations in genes already known to be associated with cancer predisposition. The experience of Nieminen et al highlights the “agony and ecstasy” of modern genetics research. NGS technologies generate massive amounts of genetic data and bioinformatics and computational strategies must continue to evolve to enable us to analyze these complex data effectively. Family studies continue to be valuable for providing a framework on which to overlay genetic data. The search for a needle in a haystack is difficult even when you have an idea of the category of gene you are looking for; however, when confronted with 1000 gene variants in each subject, any one of which might be “real,” the value of being able to compare data from affected and unaffected family members to inform variant filtering and annotation cannot be underestimated. A major question which remains is how many common highly penetrant variants are yet to be discovered. As information regarding germline and somatic variants in different diseases continues to increase, it is possible we will discover novel pathways involved in neoplasia not previously on our radar for CRC or malignancy. FCCX is most likely etiologically heterogeneous. It is almost certain that the RPS20 and CRC risk connection is private to a single family or shared by a small subset of FCCX families. At the fundamental level, this is an exciting observation because it reveals an entire new class of genes under candidacy for other types of cancer risk. ELENA M. STOFFEL Division of Gastroenterology Department of Internal Medicine University of Michigan Health System Ann Arbor, Michigan CHARIS ENG Genomic Medicine Institute and Taussig Cancer Institute Cleveland Clinic and Department of Genetics and Genome Sciences and Comprehensive Cancer Center Case Western Reserve University Cleveland, Ohio
References 1. Jasperson KW, Tuohy TM, Neklason DW, et al. Hereditary and familial colon cancer. Gastroenterology 2010; 138:2044–2058.
556
2. Vasen HF, Watson P, Mecklin JP, et al. New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC. Gastroenterology 1999; 116:1453–1456. 3. Boland CR, Goel A. Microsatellite instability in colorectal cancer. Gastroenterology 2010;138:2073–2087.e3. 4. Lindor NM, Rabe K, Petersen GM, et al. Lower cancer incidence in Amsterdam-I criteria families without mismatch repair deficiency: familial colorectal cancer type X. JAMA 2005;293:1979–1985. 5. Nieminen TT, O’Donohue M-F, Wu Y, et al. Germline mutation of RPS20, encoding a ribosomal protein, causes predisposition to hereditary nonpolyposis colorectal carcinoma without DNA mismatch repair deficiency. Gastroenterology 2014;147:595–598. 6. Comino-Mendez I, Gracia-Aznarez FJ, Schiavi F, et al. Exome sequencing identifies MAX mutations as a cause of hereditary pheochromocytoma. Nat Genet 2011;43: 663–667. 7. Jones S, Hruban RH, Kamiyama M, et al. Exomic sequencing identifies PALB2 as a pancreatic cancer susceptibility gene. Science 2009;324:217. 8. Mirabello L, Macari ER, Jessop L, et al. Wholeexome sequencing and functional studies identify RPS29 as a novel gene mutated in multi-case Diamond-Blackfan anemia families. Blood 2014; 124:24–32. 9. Palles C, Cazier JB, Howarth KM, et al. Germline mutations affecting the proofreading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas. Nat Genet 2013;45:136–144. 10. Tenesa A, Dunlop MG. New insights into the aetiology of colorectal cancer from genome-wide association studies. Nat Rev Genet 2009;10:353–358. 11. Manolio TA, Collins FS, Cox NJ, et al. Finding the missing heritability of complex diseases. Nature 2009;461: 747–753. 12. Bennett KL, Mester J, Eng C. Germline epigenetic regulation of KILLIN in Cowden and Cowden-like syndrome. JAMA 2010;304:2724–2731. 13. Nieminen TT, Abdel-Rahman WM, Ristimaki A, et al. BMPR1A mutations in hereditary nonpolyposis colorectal cancer without mismatch repair deficiency. Gastroenterology 2011;141:e23–26.
Reprint requests Address requests for reprints to: Elena M. Stoffel, MD, MPH, University of Michigan Health System, Division of Gastroenterology, 3912 Taubman Center, 1500 E. Medical Center Drive, Ann Arbor, Michigan 48109. e-mail: [email protected]. Conflicts of interest The authors disclose the following: Charis Eng is a member of the External Strategic Advisory Board of N-of-One and an unpaid member of the External Scientific Advisory Boards of Ecoeos and GenomOncology. Dr Stoffel discloses no conflicts. © 2014 by the AGA Institute 0016-5085/$36.00 http://dx.doi.org/10.1053/j.gastro.2014.07.031
