Gastroenterology 2018;154:2045–2059

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REVIEWS IN BASIC AND CLINICAL GASTROENTEROLOGYAND HEPATOLOGY

Ernst J. Kuipers and Vincent W. Yang, Section Editors

Advances in Evaluation of Chronic Diarrhea in Infants

Jay R. Thiagarajah,1 Daniel S. Kamin,1 Sari Acra,2 Jeffrey D. Goldsmith,3

Joseph T. Roland,2 Wayne I. Lencer,1 Aleixo M. Muise,4,5 James R. Goldenring,2

Yaron Avitzur,4 Martín G. Martín,6 and the PediCODE Consortium

1Division of Gastroenterology, Hepatology and Nutrition, Boston Children’s Hospital, Harvard Medical School, Boston,Massachusetts; 2Departments of Surgery and Pediatrics and the Epithelial Biology Center, Vanderbilt University School ofMedicine, Nashville, Tennessee; 3Department of Pathology, Boston Children’s Hospital, Harvard Medical School, Boston,Massachusetts; 4Division of Gastroenterology, Hepatology and Nutrition, Department of Paediatrics, The Hospital for SickChildren, University of Toronto, Toronto, Ontario, Canada; 5SickKids Inflammatory Bowel Disease Center and Cell BiologyProgram, Department of Paediatrics and Biochemistry, University of Toronto, Hospital for Sick Children, Toronto, Ontario,Canada; 6Department of Pediatrics, Division of Gastroenterology and Nutrition, Mattel Children’s Hospital and the David GeffenSchool of Medicine, University of California Los Angeles, Los Angeles, California

Abbreviations used in this paper: CODE, congenital diarrhea and enter-opathy; CTE, congenital tufting enteropathy; EEC, enteroendocrine cell;EPCAM, epithelial cell adhesion molecule; MVID, microvillus inclusiondisease; NEC, necrotizing enterocolitis; PN, parenteral nutrition; SBS,short-bowel syndrome.

Most current article

© 2018 by the AGA Institute

Diarrhea is common in infants (children less than 2 yearsof age), usually acute, and, if chronic, commonly caused byallergies and occasionally by infectious agents. Congenitaldiarrheas and enteropathies (CODEs) are rare causes ofdevastating chronic diarrhea in infants. Evaluation ofCODEs is a lengthy process and infrequently leads to a cleardiagnosis. However, genomic analyses and the develop-ment of model systems have increased our understandingof CODE pathogenesis. With these advances, a new diag-nostic approach is needed. We propose a revised approachto determine causes of diarrhea in infants, includingCODEs, based on stool analysis, histologic features,responses to dietary modifications, and genetic tests. Afterexclusion of common causes of diarrhea in infants, theevaluation proceeds through analyses of stool character-istics (watery, fatty, or bloody) and histologic features, suchas the villus to crypt ratio in intestinal biopsies. Infantswith CODEs resulting from defects in digestion, absorption,transport of nutrients and electrolytes, or enteroendocrinecell development or function have normal villi to cryptratios; defects in enterocyte structure or immune-mediated conditions result in an abnormal villus to cryptratios and morphology. Whole-exome and genomesequencing in the early stages of evaluation can reduce thetime required for a definitive diagnosis of CODEs, or lead toidentification of new variants associated with these en-teropathies. The functional effects of gene mutations canbe analyzed in model systems such as enteroids or inducedpluripotent stem cells and are facilitated by recent ad-vances in gene editing procedures. Characterization andinvestigation of new CODE disorders will improve man-agement of patients and advance our understanding ofepithelial cells and other cells in the intestinal mucosa.

Keywords: Pediatric; Gastrointestinal Disorder; Inherited;Detection.

iarrhea during early infancy (children less than 2

0016-5085/$36.00https://doi.org/10.1053/j.gastro.2018.03.067

Dyears of age) is relatively common, generally mild,and often self-limited. Most frequently, this form of diarrhea

in infancy is of allergic or infectious origin, and usually notassociated with any significant long-term sequelae. Incontrast, anatomical disorders, such as gastroschisis,necrotizing enterocolitis (NEC), or acute volvulus can causeshort-bowel syndrome (SBS) and long-lasting diarrhea aftersurgical resection.1 Another class of persistent and severediarrhea presenting in the first weeks of life results frommonogenic disorders and is termed congenital diarrhea andenteropathies (CODEs). CODEs are typically associated withfeeding intolerance and malabsorption. Both CODE and SBSrequire significant dietary and therapeutic interventions,including specialized formulas or parenteral nutrition (PN)to sustain appropriate growth, electrolyte, and nutrientbalance.

The availability of genetic diagnostic testing before 2008was limited, and infants with many of the non-surgicalsevere diarrheal disorders often carried the catch-alldiagnosis of “chronic diarrhea of unknown etiology.”These infants followed a diagnostic odyssey and clinicalcourse associated with high levels of morbidity and pro-longed expensive hospitalizations. In recent years, ourunderstanding of the underlying genetic basis of these disor-ders, as with other rare Mendelian diseases, has been revolu-tionized by the availability of next-generation sequencing.These technologies have enabled the elucidation of the geneticbasis of an increasing number of monogenic disorders causingCODEs, and most so far involve intestinal epithelial and/orimmune function. However, these disorders are rare and cleardiagnostic and therapeutic pathways for the care of thesepatients have not been established.

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Here, we outline a prioritized approach for the diagnosisand evaluation of diarrhea in infants. The approach recog-nizes recent advances in next-generation sequencing, stemcell biology, gene editing, and other aspects of the rapidlyevolving field of precision medicine.

Causes of Diarrhea During Early InfancyDiarrhea is typically defined as acute for diarrhea lasting

less than 2 weeks, or chronic when diarrhea persists formore than 2 weeks. In middle- and low-income countries, theterm persistent diarrhea is also used to denote diarrhealasting more than 2 weeks after infection or in relation toenvironmental enteric dysfunction.2,3 Historically, thenomenclature for chronic diarrhea in infancy has includedterms such as intractable or protracted diarrhea of infancy.4

Our understanding and classification of chronic diarrheas ininfants has evolved and the disease may be more helpfullydivided into either acquired diarrheas or CODEs.

Acquired diarrheas can develop postnatally or can havetheir origin in utero, with varying severity, comorbid condi-tions, and underlying pathogeneses. Postnatally acquireddisorders are most often infectious due to common entericviral, bacterial, or less commonly, parasitic pathogens, orcaused by allergic disorders induced by exposure to foodallergens, such as cow’s milk protein. Infectious causes oftenlead to acute symptoms, but in some cases can result inchronic diarrhea in immunodeficient patients or in persistentdiarrhea due to a malabsorptive enteropathy, also termedpost-infectious enteropathy.5 NEC, an important postnatallyacquired disorder, is almost always seen in premature infantsand is thought to result from gut ischemia and the influx ofpro-inflammatory lymphocytes secondary to abnormal gutmicrobial responses via Toll-like receptor 4 signaling.6–8 Thedisease can lead to SBS after gut resection. Other acquireddiarrheas leading to surgical SBS include in utero anatomicabnormalities, such as atresias and gastroschisis.

CODEs are less common but are often extremely severe,and represent unique diagnostic and management chal-lenges. Most CODEs are monogenic in nature and can bebroadly divided into genetic variants directly affecting theintestinal epithelium or variants affecting the immune sys-tem that secondarily cause severe impairment of epithelialfunction.

Diarrhea TerminologyPrevious classifications have often divided diarrhea into

osmotic and secretory, but these terms can be misleading.Therefore, some new terms are proposed.9

Osmotic diarrhea. The term osmotic diarrhea hasbeen used traditionally to refer to diarrhea resulting fromunabsorbed solutes or nutrients; however, all diarrheainvolves osmotic forces. Therefore, we prefer to use themore precise term, diet-induced diarrhea. Diet-induceddiarrhea is characterized by an elevated stool osmotic gap(>100 mOsm). Examples include glucose or disaccharidemalabsorption.

Secretory diarrhea. The term secretory diarrhea isalso imprecise. The term describes the underlying

pathophysiology of the diarrheas caused by active ionsecretion into the intestine, but it does not describe thewatery high-salt diarrheas caused by defects in intestinalsodium absorption (eg, as seen in the congenital sodiumdiarrheas and in some viral infections). Neither can the termsecretory be used to describe all the diarrheas with a lowstool osmotic gap (<50 mOsm) (Table 1), because a lowstool osmotic gap typically results from a combination ofenhanced anion-driven fluid secretion and loss of Naþ-driven fluid absorption. We prefer to use the term electro-lyte-transport–related diarrhea. Examples include congenitalchloride or sodium diarrheas.

Mixed diarrhea. Lastly, diarrhea that is obviouslyneither secretory nor osmotic, or has an intermediate stoolosmotic gap (50–100 mOsm), has been referred to as"mixed." Intermediate values for the stool osmotic gap occurfrequently and are generally caused by a combination ofdiet-induced diarrhea and electrolyte transport–relateddiarrhea resulting from different dietary intakes at thetime of testing.

Evaluation and Diagnosis of AcquiredDiarrhea in Infants

The general evaluation and diagnostic approach to in-fants with diarrhea is outlined in Figure 1. Diarrhea in in-fancy is difficult to define based on stool frequency orconsistency, as the normal range for these parameters canvary greatly. A more reliable measure is stool weight orvolume, with diarrhea defined as a stool volume of >20 g/kg/d. From a practical standpoint, the presence of diarrheacan be inferred by the deviation from the daily stool patternand by the level of dehydration and severity of electrolyteabnormalities.

EvaluationDiarrhea presenting early or immediately postnatally in

term or premature infants should prompt evaluation forcongenital enteropathies, NEC, or anatomical abnormalities(Figure 1). For less-severe diarrhea presenting later in in-fancy, the initial workup should focus on investigatingcommon acquired etiologies, such as infections, cow’smilk protein allergy, or food protein–induced enterocolitissyndrome (Figure 1).

In both high-income and middle-/low-income countries,viral pathogens are the most likely agents responsible forinfectious diarrhea in infants. Rotavirus, cytomegalovirus,adenovirus, and norovirus should all be considered. Lesscommon in high-income countries but a significant factorglobally are enteric bacterial pathogens, such as Salmonellaenterica, Shigella spp, Campylobacter jejuni, and pathogenicEscherichia coli.2,10 Persistent viral diarrheas, or diarrheaafter administration of the rotavirus vaccine, may be thefirst sign of primary immunodeficiency or related to auto-immune enteropathies, such as immunodysregulation pol-yendocrinopathy, enteropathy X-linked.11 Bloody diarrheawith no evidence of infection should be followed by either achange to extensively hydrolyzed formula and/or amaternal dairy exclusion diet for a potential diagnosis of

Table 1.Stool Testing

Stool test Notes

Electrolytes This is a key test in evaluating the nature of the diarrhea and for calculation of the stool osmotic gap. High Naþ orCl– is often reflective of alterations in intestinal ion transport.

Stool osmotic gap calculation: 290 – 2 � (Stool Naþ þ Kþ):Stool osmotic gap >100 mOsm is defined as high; <50 mOsm is defined as low; and a gap between 50 and 100

mOsm is defined as intermediate.Osmolality In almost all cases, stool osmolality is iso-osmolar to serum (w290 mOsm) and, therefore, generally does not

need to be sent. If there is a suspicion of improper collection (delay or contamination with urine/water),measurement of stool osmolality can provide objective evidence for this, as it will be relatively dilute or hypo-osmolar to serum.

Reducing substancesand pH

Reducing substances >0.5% indicates malabsorption of monosaccharides. Low pH (<5.3) results from anabundance of short-chain fatty acids that are the products of fermentation and is indicative of carbohydratemalabsorption. However, these must be interpreted in the context of the diet, as some amount ofcarbohydrate malabsorption is normal in neonates and related to ongoing development of the intestine.

a1-antitrypsin Serum protein that is largely resistant to the action of intestinal proteases unless it is secreted in the stomach.Elevation in stool reflects intestinal protein loss (ie, protein-losing enteropathy).

Fat Quantitative 72-h fecal fat collection is ideal to evaluate fat malabsorption, although it can be difficult in practice.It requires an accurate estimate of the daily enteral fat intake to calculate daily percentage absorption.Percentage fat absorption varies with age and is relatively low in infants, with 15%–20% loss in normal stools.Qualitative or spot fecal fat (neutral or split) is a helpful initial test. Elevation in neutral fat reflects increasedmono-, di-, or triglyceride content in the stool and can suggest pancreatic insufficiency. Elevation in split fatreflects increased free fatty acids in stool and correlates with intestinal fat malabsorption

Elastase Elastase is unchanged by intestinal proteases and, if low, can imply pancreatic insufficiency, but may be dilutedand reduced in high-volume chronic diarrhea not associated with pancreatic insufficiency.

Occult blood Microscopic blood can be evaluated in the stool, however, it should be noted that it is unhelpful as a discriminatorof bowel inflammation vs cutaneous irritation caused by severe diarrhea. We therefore do not recommend itsuse in initial testing.

Inflammatory markers Elevated lactoferrin or calprotectin are well-correlated with the presence of intestinal inflammation.Infection studies Standard assessment of enteric bacterial pathogens by culture and/or by polymerase chain reaction. Viral

pathogens, such as rotavirus, cytomegalovirus, norovirus, and adenovirus, should be tested by polymerasechain reaction and/or enzyme-linked immunosorbent assay.

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cow’s milk protein colitis.12 Food protein–induced entero-colitis syndrome, a primarily T-cell–mediated inflammatorydisorder triggered by a variety of food antigens, can presentin infancy with acute-onset diarrhea, usually after dietaryintroduction of solid foods.13,14

Evaluation of severe early-onset diarrhea by radiographicsmall-bowel follow-through studies should be used to iden-tify intestinal malrotation with intermittent volvulus, orcongenital short gut. Hirschsprung’s disease can rarely pre-sent with diarrhea, and should also be considered. Obstruc-tive symptoms without an identifiable lead point are typicallyassociated with chronic intestinal pseudo-obstruction. NECcan present with watery or bloody diarrhea, usually in as-sociation with prematurity, abdominal distension, feedingintolerance, and temperature instability. Initial evaluationand management involves serial abdominal films to investi-gate the presence of bowel wall gas, cessation of enteralfeeding, and antibiotic administration.6,15

Evaluation of SuspectedMonogenic DiarrheaEvaluation

Increased suspicion for CODE can be gleaned from thehistory and clinical course. Important information to gatherincludes prenatal history and testing; age at onset of

symptoms; nature of symptoms; extraintestinal manifesta-tions; nutrition and diet history; and a full family history,including any evidence of consanguinity and ethnicity(Figure 1). Specific populations have relatively high incidencerates of congenital enteropathies, such as the Finnish,Ashkenazi Jews, Navajo Native American, and those origi-nating from the Arab Gulf regions.16–19

In patients undergoing evaluation after 1 month of age,the age of onset of the diarrhea is a key historical detail,with very-early-onset increasing the chance of CODE. In-fectious and immune-related conditions generally have asymptom-free postnatal period of at least a few weeksbefore clinical symptoms are apparent. It should be notedthat, in some conditions, the volume of diarrhea may be sosevere that less-experienced parents and health care pro-viders may confuse diarrhea for urine, delaying recognitionand appropriate consultation.

Serum TestingInitial testing should include a complete blood count,

serum electrolytes, inflammatory markers, liver functiontests, immunoglobulin levels, a lipid panel (triglycerides,cholesterol), fat-soluble vitamins, a coagulation profile, andzinc level. Additional immunologic investigation should beconsidered if there is suspicion of immune dysfunction, suchas specific T- and B-cell subset analysis.

Figure 1. Initial evaluationof neonate/infant withdiarrhea. Initial evaluationof diarrhea in infantsstarting with key historicalinformation, identificationof red-flag findings, andkey stool studies andblood tests. Infants withred-flag findings or thatrequire critical interventionsoon after birth will needevaluation for NEC andanatomical abnormalities,as well as further investi-gation for congenitaldiarrheas. Important com-mon etiologies to investi-gate especially presentingbeyond the neonatalperiod include cow’s milkprotein colitis, infections,food protein enterocolitis.CBC, complete bloodcount; CMV, cytomegalo-virus; CRP, C-reactiveprotein; ESR, erythrocytesedimentation rate; GI-SBFT, gastrointestinalfluoroscopy with smallbowel follow-through,NPO, nil per os.

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Stool TestingAccurate stool testing is a key component of the diag-

nostic evaluation for congenital diarrheas. Collection of stoolsamples can be challenging due to the presence of mixedurine and stool in the diapers of neonates, and by rapid lossof stool water content due to absorption into the diapermaterial. Use of a urine catheter for a few days may allowfor accurate stool sampling and volume measurement.Ideally, fresh stool samples obtained immediately afterexcretion should be tested (Table 1). A key component inthe evaluation of stool testing is an exact quantification ofdietary intake at the time of testing.

Diagnostic Algorithm for Suspected MonogenicDiarrheas and Enteropathies

The workup for CODE is initiated after the exclusion ofacquired diarrhea. As an initial diagnostic evaluation, it ishelpful to grossly characterize the stool appearance in sus-pected CODE (Figures 1 and 2). Although sometimes diffi-cult, most stools can be broadly differentiated into 3 maincategories: watery, fatty, and bloody stools, and this allowsfor clear prioritization of initial testing. Watery stool ischaracterized by a high liquid content—often with very littleform—that can be mistaken for urine. Fatty stools areusually foul-smelling, can have a bulky or “fluffy” appear-ance, are pale in color, and/or are spot fecal fat–positive.Bloody stools contain gross blood mixed in with stools. The

presence of large volumes of bright red blood or melenashould trigger evaluation of vascular or anatomic gastroin-testinal bleeding as well as infection.

It should be noted that genetic testing (see sectionGenomic Testing) can occur in parallel or early in thediagnostic algorithm, especially if there are clear factors tosuspect a monogenic diarrheal disease, such as significantconsanguinity; a family history of gastrointestinal disease ininfancy; and clinical indicators, such as diarrhea severityand neonatal onset.

Evaluation of Watery DiarrheaAfter documentation of diarrheal output with normal

feeding, evaluation should include a no-feeding (nil per os)trial of at least 24 hours, with assessment of stool output andelectrolytes (Figure 2, top). If diarrheal volume is unchangedor is minimally changed after fasting, this points to electro-lyte-transport–related diarrhea. Significant improvement indiarrheal output after termination of enteral feeds pointsto diet-induced diarrhea. Subsequent evaluation should aimto elucidate whether a specific nutrient is malabsorbed or ifthe patient has a generalized malabsorptive diarrhea.A feeding trial with carbohydrate-free (Ross Carbohydrate-Free; Abbott, Macquarie Park, NSW, Australia) or fructose-based formula (Galactomin-19; Nutricia, Wiltshire, UK)leading to significant improvement, along with reducedstool pH and elevated stool-reducing substances with

Figure 2. Diagnostic algo-rithm for evaluation ofCODEs. Abetalipo, abetali-poproteinemia; CLD,congenital lactase defi-ciency; CMP, cow’s milkprotein; DGAT1, diac-ylglyceroltransferase 1;GGM, glucose galactosemalabsorption; Hypo-betalipo, hypobetali-poproteinemia; NEUROG3,Neurogenin3 deficiency;pBAD, primary bile aciddiarrhea; BAM, bile acidmalabsorption; PCSK1,proprotein convertasekinase deficiency; RD,retention disease; Suc/Iso,sucrose-isomaltase defi-ciency; TTC7A, tetra-tricopeptide repeat domain7A; VEOIBD, very-early-onset inflammatory boweldisease.

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carbohydrate-containing formula, supports the diagnosis ofcarbohydrate malabsorption. Elucidating the specific abnor-mality of carbohydrate assimilation can be performed withspecific monosaccharide (glucose and fructose), disaccharide(sucrose, lactose, and maltose) dietary challenges andassessed by changes in stool volume and/or breath hydrogentesting.20 Disaccharidase activity assays for lactase, sucrase,maltase, and palatinase performed on proximal small bowelbiopsies may be helpful to diagnose disaccharidase defi-ciency.21 However, these enzymatic assays are often unreli-able due to poor sampling or in the setting of inflammationor villus atrophy due to secondary disaccharidase deficiency.

If there is no clear evidence of a selective carbohydratemalabsorption, diagnostic evaluation should begin withesophagoduodenoscopy and flexible sigmoidoscopy withbiopsies for histologic analysis; if not contraindicated by theclinical status of the infant.22 Biopsies should include sam-ples for both routine histology and electron microscopy, aswell as for measuring mucosal disaccharidase activity.

Early endoscopy and biopsy differentiating normal froman abnormal villus to crypt ratio,23 and/or an inflammatorypredominance allows for significant streamlining and pri-oritization of the evaluation, planning for dietary in-terventions, and initiation of genetic testing.

Parallel assessment for a possible protein-losingenteropathy is important, as it may be indicative of acompromised epithelial barrier, suggesting autoimmuneenteropathy, or newly described monogenic disorders, suchas DGAT1 and CD55 deficiency. The presence of elevatedlevels of a1-antitrypsin in the stool and low serum albumin,IgG, and lymphopenia are consistent with protein-losingenteropathy.24,25

Evaluation of Fatty DiarrheaEvaluation of fatty diarrhea is by spot fecal fat testing,

including neutral and split fat and, if available, quantita-tively by 72-hour stool fat collection (Figure 2). Stool elas-tase is a useful initial test, as it can help distinguish betweenconditions resulting from pancreatic insufficiency and thosecaused by intestinal fat malabsorption, although fecal elas-tase can often be falsely low (false positive) with high-volume diarrhea. The presence of fat-laden enterocytes inhistologic sections along with serum lipid abnormalities canpoint to disorders of fat transport and metabolism, such aschylomicron retention disease and abetalipoproteinemia.Pancreatic insufficiency is confirmed by the responsivenessof diarrheal symptoms to enzyme replacement therapy.26

Evaluation of Bloody DiarrheaThe presence of gross blood implies significant colitis

and further evaluation should include stool inflammatorymarkers and endoscopy (Figure 2). The presence of in-flammatory changes in histologic sections should precipitatefurther investigation of infantile very-early-onset inflam-matory bowel disease, autoimmune enteropathy, or primaryimmunodeficiency. It should be noted that not all forms ofimmune-dysregulation–related enteropathies are associatedwith bloody stools.27

Endoscopy/HistologyInitial evaluation of H&E-stained sections should focus

on overall intestinal epithelial architecture, namely villus tocrypt ratio, abundance of the epithelial cell type and

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structure, and the immune cell composition in the laminapropria and intraepithelial compartments.28

� Normal villus/crypt architecture: Suggests conditionswith defective digestion, absorption and transport ofnutrients and electrolytes, or defects of enter-oendocrine cells.

� Abnormal villus/crypt architecture: Villi are generallyshort or flattened and may be associated with eithercrypt hyper- or hypoplasia, and may be seen in defectsof enterocyte structure, vesicular trafficking, differenti-ation, and immune-mediated conditions. Specifically,villus blunting and apoptosis at the crypt base is sug-gestive in most cases of an immune-mediated disorder.Epithelial crowding and disorganization at the top ofthe villus is found in disorders of adhesion, such ascongenital tufting enteropathy (CTE), and is associatedwith crypt hyperplasia.

� The relative abundance and distribution of cells in thestem cell–based compartment and differentiated cells ofthe epithelium can be appreciated by H&E and by cell-type–specific staining. Selective depletion of differenti-ated cell types may be associated with autoimmuneenteropathy and endocrinopathies.

� Abnormal abundance or absence of mononuclear cellsin the lamina propria and intra-epithelial compartmentssuggest an immune-mediated disorder. A dominance ofeosinophilic infiltrates within the mucosa-associatedwith villus blunting may be an indicator of eosino-philic gastroenteritis.

Immunohistochemical AssessmentsImmunohistochemical staining of differentiated cell

populations may be helpful to confirm H&E findings and/ora specific diagnosis (Figure 3, Supplementary Table 1).Staining may be limited by the small amount of availabletissue acquired during endoscopy. The assessment includesstaining for enteroendocrine (chromogranin/synaptophy-sin), Paneth (lysozyme), and goblet cells (periodic acid–Schiff). Identification of proliferating cells can be done usingKi67, proliferating cell nuclear antigen, or pHisH3. Immunecell types can be initially assessed using specific surfacemarkers for B cells (CD20), T cells (CD3/CD4/CD8), mac-rophages (PU.1/CD68/F4/80), and plasma cells (CD138);however, identification of specific immune cell subsets re-quires more extensive and specialized staining.

If histologic assessment suggests abnormal epithelialarchitecture, initial immunohistochemical staining in allcases should include CD10/villin (microvillus inclusiondisease [MVID]), periodic acid–Schiff (DGAT1), MOC31(CTE), and frozen-section staining with Oil Red O if lipidtrafficking disorders are under consideration.29,30

Further evaluation includes electron microscopy toassess the presence and relative size and location of themicrovilli and to identify intracellular microvillus inclusionsor abnormal vesicular structures suggestive of disorders ofintracellular trafficking.

Immunolocalization of specific transporter, structural, orintracellular proteins, such as (DGAT1, PCSK1, and others)may provide increased diagnostic validation. Figure 3 de-picts the typical histology of MVID, CTE (epithelial celladhesion molecule [EPCAM]), abetalipoproteinemia, andautoimmune enteropathy (immune-dysregulation–relatedenteropathies). All studies, however, should be performed inconcert with genetic analyses.

Genomic TestingAdvances in next-generation sequencing technologies

promise to shorten the diagnostic odyssey for many CODEpatients. Although the clinical diagnostic algorithm providesa framework for evaluation and prioritized testing, in manycases where the diagnosis of CODE is highly suspected butthe specific etiology is not identified or requires confirma-tion, either targeted genetic testing (Sanger sequencing) orwhole-exome sequencing can identify the genetic cause andallow for appropriate early treatment.

In selective populations with a high prevalence of knownspecific genomic variants, or when the diagnostic evaluationis strongly suggestive of a specific disorder, such as thecharacteristic epithelial tufts seen on biopsy in EPCAM mu-tations, Sanger sequencing should be considered for rapiddiagnosis and treatment. For example, there are a number ofrelatively common founder CODE gene mutations, includingMexican/Arab: EPCAM (c.491þ1G>A and c.498insC),18,31

Ashkenazi Jews: DGAT1 (IVS8þ2 T>C);32 Navajo: MYO5B(p.Pro660Leu),33 and Finns: SLC26A3 (p.Val317del).34

In cases of a suspected CODE, where the diagnosisbased on clinical evaluation is unclear, it is now standard ofcare to perform whole-exome sequencing to identify apossible causative genetic mutation.35–38 It should alwaysbe considered, however, that whole-exome sequencing maynot detect genetic defects in genes with poor coverage,large insertions and deletions, and mutations in regulatoryand splice or intronic regions. Therefore, in certain caseswith a high likelihood of a monogenic disorder, whole-genome sequencing, or RNA sequencing should also beconsidered. Microarray comparative genomic hybridizationis a rapid and frequently used method to assess significantcopy number variation and changes in whole chromo-somes, and discovery of large deletions (>200 kb) andduplications.39

Classification and Pathophysiologyof Diarrhea in Infants: AcquiredDiarrheal Disorders

SBS occurs more frequently in premature neonates (25/100,000 live births) and is associated with either anatomicalintestinal defects or NEC. Many of these neonates will havemalabsorption early on and be dependent on PN to sustainnormal growth and development.1,40

Disorders such as gastroschisis41 may result in diarrheaby various mechanisms, including impaired motility,42 sur-gical formation of ostomies that shorten bowel length, orbacterial overgrowth. Patients with gastroschisis can also

Figure 3. CODE histology. (A) Autoimmune enteropathy (H&E) showing extensive villus atrophy, crypt hyperplasia, increasedlamina propria inflammation (arrows), neutrophilic crypt abscess (asterisk). Scale bar ¼ 50 mm. (B) Autoimmune enteropathy(H&E) showing crypt base apoptosis. Scale bar ¼ 10 mm. (C) Autoimmune enteropathy (H&E) showing crypt intraepitheliallymphocytosis, absent Paneth cells and rare crypt epithelial apoptosis (arrows). Scale bar ¼ 10 mm. (D) Abetalipoproteinemia(H&E) showing fat globules in enterocytes. Scale bar ¼ 25 mm. (E) Normal villus stained with villin showing clear brush-borderstaining (arrows). Scale bar ¼ 5 mm. (F) MVID patient with abnormal villin staining with villin signal present in intracellularmicrovillus inclusions (arrows) and relative lack of brush-border staining (asterisk). Scale bar ¼ 5 mm. (G) MVID patient withabnormal intracellular CD10 staining (arrows). Scale bar ¼ 10 mm. (H) Transmission electron micrograph showing characteristicmicrovillus inclusion in MVID. (I) Tufting enteropathy patient showing characteristic epithelial tufts in the small intestine(arrows). Scale bar ¼ 25 mm. (J) Membranous MOC31 (EPCAM) staining in unaffected patient. Scale bar ¼ 50 mm. (K) Completeloss of MOC31 (EPCAM) staining in tufting enteropathy patient. Scale bar ¼ 50 mm. (L) Characteristic tufts also seen in thecolon in tufting enteropathy (arrows). Scale bar ¼ 25 mm.

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acquire SBS as a result of volvulus that can occur before orafter birth.

Intestinal atresia results from defects in intestinaldevelopment early in gestation due to vascular anomaliesor inherited defects in luminal development.43 Atresia canbe single, with reasonable complement of distal bowel, orthere can be multiple atresias, with very limited bowellength. Next-generation sequencing has resulted in theidentification of several severe inherited forms of intes-tinal atresia, including TTC7A and RFX6 (discussedDisorders of Epithelial Trafficking and Polarity).44,45

NEC is predominantly an illness that affects preterminfants who are initiating enteral feeding. The pathogenesisis not clearly defined, but includes vascular abnormalities,bacterial dysbiosis, and abnormal immune responses, withthe final common pathway being intestinal ischemia.7 Thiscan vary in severity, but uncommonly can include gangreneof nearly the entire small and large intestines. Diagnosiscurrently relies on careful clinical observation and plainfilms. Early biomarkers have been investigated, but are

not yet used in routine clinical practice.46 As with theaforementioned conditions, diarrhea and nonselectivemalabsorption result from SBS.

Monogenic Diarrheal DisordersMonogenic diarrheal disorders can be broadly classified

into 5 major categories (Figure 4, Table 2) reflective of acommon pathophysiology, although there remains overlapamong a number of these categories.47 Epithelial cell defectsare the hallmark of the first 4 CODE categories and rangefrom defects in epithelial transporters, enzymes, and meta-bolism to defects in epithelial trafficking and polarity andenteroendocrine cell dysfunction. The clinical presentation isalmost always within the first several months of life and isassociated with high-volume watery diarrhea.

The fifth category encompasses monogenic disordersthat cause dysfunction of the immune system, which resultin a wide spectrum of both intestinal and extraintestinalmanifestations. This broad array of monogenic entities

Figure 4. Pathophysiology of CODEs: Illustration of major categories of CODEs with example disorders. (A) Disorders ofepithelial transport: GGM, glucose galactose malabsorption affecting sodium glucose cotransporter SLC5A1; CCD, congenitalchloride diarrhea affecting the Cl/HCO3

– exchanger DRA (SLC26A3); CSD, congenital sodium diarrhea affecting the Na/Hþexchanger, NHE3 and the guanylin receptor GC-C (GUCY2C); cGMP, cyclic guanosine monophosphate. (B) Disorders ofepithelial enzymes and metabolism: SI, sucrase-isomaltase deficiency; LCT, lactase deficiency; DGAT1, diacylglycerol-transferase 1 deficiency, hypobetalipoproteinemia affecting apolipoprotein B (ApoB), abetalipoproteinemia affecting micro-somal triglyceride transfer protein (MTTP) and chylomicron retention disease affecting SAR1B. (C) Disorders of epithelialtrafficking and polarity: MVID affecting myosin 5b (MYO5B) and syntaxin 3 (STX3), Tufting enteropathy affecting EPCAM.TTC7A, tetratricopeptide repeat domain 7A. (D) Disorders of enteroendocrine cells: PCSK1, proprotein convertase kinasedeficiency; NEUROG3, Neurogenin3 deficiency; RFX6, Mitchell-Riley syndrome. (E) Immune dysregulation–associated en-teropathy: X-linked inhibitor of apoptosis (XIAP) affecting BIRC4, FOXP3, CTLA4, LRBA affecting T- and B-cell regulation/stimulation.

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Table 2.Classification of Diarrheas and Enteropathies in Infants

Variable

Gene

Protein functionName OMIM no. Inheritance

Epithelial nutrient/electrolyte transportCongenital chloride diarrhea SLC26A3 126650 AR Cl–/HCO3

–exchangerCongenital sodium diarrhea SLC9A3 616868 AR Naþ/Hþ exchanger (NHE3)Congenital sodium diarrhea GUCY2C 601330 AD Guanylate cyclaseGlucose-galactose malabsorption SLC5A1 606824 AR Naþ-glucose cotransporterPrimary bile acid diarrhea SLC10A2

SLC51B601295 AR Ileal bile salt transporters

Acrodermatitis enteropathica SLC39A4 201100 AR Zinc transporterEpithelial enzymes and metabolism

Congenital lactase deficiency LCT 603202 AR DisaccharidaseSucrase-isomaltase deficiency SI 609845 AR DisaccharidaseTrehalase deficiency TREH 612119 AR DisaccharidaseEnterokinase deficiency TMPRSS15 606635 AR Pro-enterokinaseDGAT1 deficiency DGAT1 604900 AR Triglyceride synthesisPLVAP deficiency PLVAP 607647 AR Endothelial fenestraeAbetalipoproteinemia MTP 157147 AR Microsomal triglyceride transfer proteinHypobetalipoproteinemia APOB

ANGPTL3107730605019

ARAR

Lipid absorption

Chylomicron retention disease SAR1B 607690 AR Intracellular chylomicron traffickingDyskeratosis congenita TERT 613989 AR/AD Maintenance of telomeresKabuki syndrome KMT2D 147920 AD Histone methyltransferase

Epithelial trafficking and polarityMicrovillus inclusion disease MYO5B

STX3606540600876

ARAR

Cellular trafficking, polarity, and signaling

Tufting enteropathy EPCAM 185535 AR Cell–cell adhesion and signalingSyndromic Naþ diarrhea SPINT2 605124 AR Serine protease inhibitorTrichohepatoenteric syndrome 1 TTC37 614589 AR Cell polarity and signalingTrichohepatoenteric syndrome 2 SKIV2L 600478 AR HelicaseFamilial hemophagocytic lymphohistiocytosis 5 STXBP2 613101 AR Syntaxin-binding proteinTTC7A deficiency TTC7A 609332 AR Protein transport and trafficking

Enteroendocrine cell dysfunctionEnteric anendocrinosis NEUROG3 604882 AR Transcription factor–cell fateX-linked lissencephaly and MR ARX 300382 X-linked Homeodomain transcription factorsProprotein convertase 1/3 deficiency PCSK1 162150 AR Neuroendocrine convertaseMitchell-Riley syndrome RFX6 612659 AR Transcription factor–cell fate

Immune dysregulation–associated enteropathya

IPEX FOXP3 300292 X-linked Regulatory T-cellICOS deficiency ICOS 604558 AR T-cell receptorADAM17 deficiency ADAM17 603639 AR TNF-a–converting enzymeEGFR deficiency EGFR 616069 AR EGF receptorCD55 deficiency CD55 125240 AR Complement cascade inhibitorCTLA4 deficiency CTLA4 123890 AD Costimulatory signalingLRBA deficiency LRBA 606453 AR Endosomal trafficking regulatorXIAP BIRC4 300079 X-linked Inhibitor of apoptosis

AD, autosomal dominant; AR, autosomal recessive; MR, mental retardation; OMIM, Online Mendelian Inheritance in Man.aFor a detailed list of very-early-onset inflammatory bowel disease/autoimmune enteropathy genes refer to Uhlig et al.75

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includes genetic defects also classified as infantile-onsetinflammatory bowel disease (children less than 2 years ofage), autoimmune enteropathy, or primary immunodefi-ciency.48 For a detailed description for the more commonspecific disorders, please see the Supplementary Material.

Disorders of Epithelial Nutrient/ElectrolyteTransport

Alterations in epithelial transport proteins representsome of the most prevalent and well-known of the

congenital diarrheas (Figure 4A). These encompass variantsof pure electrolyte transporters, such as the Cl/HCO3

–

exchanger DRA (SLC26A3), which result in congenital chlo-ride diarrhea34; the Naþ/Hþ exchanger NHE3 (SLC9A3),which results in congenital sodium diarrhea; andelectrolyte-nutrient co-transporters, such as SGLT1(SLC5A1),49 which result in loss of both sodium and glucoseabsorption. Alterations in regulatory proteins, such as theguanylin receptor GC-C (GUCY2C)50 or other transporters,such as those mediating intestinal sodium-coupled bilesalt re-uptake (SLC10A2),51 or primary excessive bile acid

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production, can induce secondary electrolyte transport de-fects in epithelial cells and excessive fluid loss.52 Thesedisorders generally exhibit a structurally intact epitheliumand brush border with a normal villus to crypt ratio.Excessive stool chloride levels are found in congenitalchloride diarrhea (SLC26A3)53 whereas high stool sodiumlevels are found in congenital sodium diarrhea (SLC9A3,GUCY2C, SPINT2). Both of these disorders usually presentimmediately at birth with polyhydramnios often present inutero. In contrast, glucose-galactose malabsorption exhibitsa diet-induced dehydrating diarrhea initially evident afterinitiation of feeding that ceases after institution of a glucose-galactose free diet.

Disorders of Epithelial Enzymes and MetabolismAlterations in a number of important enzymes involved in

both nutrient absorption as well as epithelial cell metabolismresult in severe diarrhea (Figure 4B). Defects in brush-borderenzymes involved in carbohydrate digestion, such as lactaseand sucrose-isomaltase, result in a diet- induced diarrheawith onset after intake of carbohydrate-containing formula orfood. These include the relatively common lactose intolerancecaused by reduced function of LPH,54,55 which also may beacquired after gastroenteritis or due to prematurity, vs thevery rare cases of total loss-of-functionmutations in LPH56 orin the gene for sucrase-isomaltase (SI).57 Bi-allelic mutationsof SI result in a loss of sucrose or isomaltase, or both enzymeactivities, and will result in diarrhea on a diet containingsucrose and/or starch, isomaltose, and maltose. Disorders ofthese brush-border enzymes exhibit grossly normal intesti-nal histology after biopsy.

A more recently described CODE characterized by anelectrolyte transport–related diarrhea, emesis, protein-losingenteropathy, and growth failure induced by enteral intake oflipids was found to be due to a loss-of-function mutation inDGAT1, which is involved in cellular triglyceride formation.32

Initial studies have indicated some loss in brush-bordermicrovillus structure in patient biopsies, although it is un-clear whether this persists in the absence of enteral lipids.

Other disorders of fat transport or metabolism resultfrom mutations in proteins involved in fat absorption acrossthe epithelium, such as microsomal triglyceride transferprotein resulting in abetalipoproteinmeia, apolipoprotein Bresulting in hypobetalipoproteinemia, or chylomicronretention disease (SAR1B). These disorders classically showlipid-laden vacuoles within enterocytes (Figure 3D).

Disorders of Epithelial Trafficking and PolarityA number of disorders of epithelial trafficking and po-

larity lead to early-onset diarrhea, usually appearing in thefirst months of life. All disorders carry an autosomalrecessive inheritance and have been described in variousethnic groups. The diagnosis is based on typical pathologicfindings and/or extraintestinal manifestations, followed byconfirmation of the diagnosis via genetic testing. The twomost well-described disorders are MVID and CTE.

MVID results from mutations in the cytoskeletal motorprotein Myosin 5b (MYO5B), which results in defective

apical membrane recycling in intestinal epithelial cells.33,58

This results in the pathognomic structural abnormalitiesof the epithelial apical membrane, including loss of micro-villi59 leading to abnormal CD10 and villin staining of in-testinal biopsies and intracellular microvillus inclusionsseen in electron micrographs (Figure 3E–H).59 Patientspresent with profuse dehydrating diarrhea in the absence ofenteral intake but worsened with feeding. A similar butmilder phenotype to MYO5B mutations can be also foundafter loss of the trafficking protein syntaxin 3 (STX3).61

CTE results from loss of function in the epithelialsignaling and adhesion protein EPCAM and causes a severesodium-losing diarrhea that usually presents from birth to 3months of life.31,62 The diarrhea in CTE does occur in theabsence of feeding, however, it is generally much worsewith enteral intake. The classical findings on biopsy includethe presence of pathognomic surface epitheial “tufts” seenon H&E stains of biopsies, as well as the lack of EPCAM(MOC31) immunostaining (Figure 3I–L).

Other disorders involving epithelial structural defectsinclude recently described mutations in the gene TTC7A,44

which leads to loss of the apicobasal polarity of the enter-ocyte, crypt-base apoptosis, crypt and villus atrophy andchronic inflammation, and TTC37 mutations,63 which resultin mild to severe villus atrophy and variable inflammatoryinfiltrate on biopsy.

Disorders of Enteroendocrine Cell FunctionDisorders classified as enteric endocrinopathies result

from either a loss of proper enteroendocrine cell (EEC) fateor generalized defects in processing of gut hormones.Collectively, these disorders result in a generalized mal-absorptive diarrhea that requires PN for the first severalyears of life, although diarrheal symptoms persist perhapsindefinitely. Each disorder is also associated with a uniqueset of systemic endocrinopathies that allow for anticipatoryguidance of physicians and families alike.

The first disorder described involving enteroendocrinedysfunction resulted from bi-allelic loss-of-function muta-tions of Neurogenin3, a basic helix–loop–helix transcriptionfactor, required for enteroendocrine and b-cell develop-ment.64,65 Patients present with a primarily diet-induceddiarrhea that is not specific to any single nutrient andtheir intestinal biopsies reveal a normal crypt to villus ratio,with selective loss of all types of EECs.

Another EEC cell disorder was found to result from loss-of-function mutations of PCSK1, coding for prohormoneconvertase (PC1/3), a protease that is required for thebiosynthetic processing of hormone precursors into theirfully functional forms.66 Infants present with diarrhea thatis phenotypically similar to NEUROG3 mutations but asso-ciated with a wider range of systemic endocrinopathies,including adrenal insufficiency, hypothyroidism, and dia-betes insipidus–like picture.

Other EEC-related disorders include mutations inRFX6,67 a transcription factor that functions both up- anddownstream of Neurogenin3 and mutations in ARX, a ho-meobox transcription factor that results in selective reduc-tion of GLP-1 and cholecystokinin-expressing EECs.68,69

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Immune Dysregulation–Associated EnteropathiesRecent genetics studies have rapidly increased the

number of monogenic disorders, including FOXP3,70,71

ICOS,72 IL10R,73 TRIM22,74 and ARPC1B,38 which causedysregulation of the immune system and subsequentlyinflammation and enteropathy in the intestine. Immune-mediated intestinal disorders present with a wide varietyof manifestations, but all have bloody or watery diarrhea,and are frequently associated with systemic disease andmulti-organ involvement. Many of these disorders havebeen classified as infantile-onset inflammatory bowel dis-ease, and have features characteristic of later-onset in-flammatory bowel disease, such as bloody diarrhea, colitis,perianal disease, and ulceration, for example, IL10R.73,75

Others have predominantly villus atrophy without ulcera-tion and present with profuse watery diarrhea and malnu-trition, for example, ICOS and FOXP3, and have also beentermed autoimmune enteropathies. Most of these diseasesare curable with bone marrow transplantation.

One of the disorders that presents with primarily waterydiarrhea without bleeding or perianal disease is immuno-dysregulation polyendocrinopathy, enteropathy X-linkedsyndrome, which results from mutations in the FOXP3gene.70,71 The intestinal pathologic features vary from com-plete villus atrophy with apoptosis in a graft-vs-hostappearance, to loss of goblet and Paneth cells, with mildinflammation. Similarly, mutations in the gene encodingICOS,72 a T-cell co-stimulatory protein, presents with waterypredominantly diet-induced diarrhea starting after a fewmonths of life with biopsy findings of villus atrophy, cryptapoptosis, and an inflammatory infiltrate (Figure 3A–C).

Functional Testing of UnknownGenetic Variants

Whole-exome/genome sequencing has led to a plethoraof new or previously unconfirmed gene mutations associ-ated with CODE. After confirmation of a variant in a novelgene, the next critical step is to confirm a plausible biolog-ical mechanism through functional assessment of genefunction. This requires careful phenotyping of patient’s cells,physiological assays of cellular function in both model sys-tems and patient-derived tissue and cells. Hints at genefunction may first be discerned from the clinical andmorphologic data acquired during the initial stages ofdiagnosis and treatment, and can direct initial tissue andcellular phenotyping.

Initial phenotyping can be carried out through charac-terization of key epithelial structural, transport, and cellulartrafficking proteins in patient-derived tissue sections.However, complete analysis of immunostaining is oftenlimited by the small amount of paraffin-embedded biopsytissue available. This characterization has been facilitated bythe advent of multiplex immunofluorescence imaging plat-forms,76 where a single section can be used to stain with upto 50 antibodies through iterative staining and imagingprocedures (Supplementary Figure 1A). These methodshave the ability to provide a detailed phenotyping of

structural defects and barrier function; epithelial cell po-larity; patterns of epithelial, immune, and neural cell dif-ferentiation; and identification of immune cell subsets.

Recent advancements in deriving intestinal organoidsfrom human induced pluripotent stem cells and isolatingmultipotent intestinal stem cells from human crypts hasprovided unprecedented opportunities to model CODEdisorders in a dish.77–80 Patient or CRISPR/Cas9-generatedintestinal stem cells or induced pluripotent stem cellsalso provides opportunities to perform high-throughputassays with intestinal epithelium. Specifically, functionalanalysis of barrier function using live intestinal enteroidsplated on filter supports in 2-dimensional formats can beachieved using electrical and biochemical measures of pas-sive intercellular and transcellular transport of small solutes(ions and membrane impermeant small solutes). Both 2-and 3-dimensional enteroid cultures can be used forassessment of active (energy-dependent) trans-epithelialion and solute transport using fluorescence ratio-imaging,enteroid swelling, and in some cases electrical approaches(Supplementary Figure 1B).

Many monogenic causes of the CODE primarily affectepithelial structure and the organization of plasma andintracellular membrane compartments. These genes affectmembrane trafficking, which can be effectively and compre-hensively assessed in enteroids and cells using assays for IgGintracellular transport by the Fc-receptor, FcRn, and trans-ferrin transport by the rapidly recycling transferrin recep-tor.81 Both probes assess function for all endosomalcompartments adapted to and serving the polarized epithelialcell phenotype (Supplementary Figure 1C).

Discussion and ConclusionsInfants with CODE pose a clinical challenge that requires

a structured diagnostic approach to allow early and correctdiagnosis. The relatively uniform presentation of early se-vere diarrhea with food intolerance and the large number ofpotential etiologies often makes it difficult to prioritize in-vestigations. The updated algorithm in this article harnessesthe recent progress in the understanding of the pathogen-esis of these disorders, along with advances in genetictesting and immunohistochemistry techniques. The algo-rithm suggests an initial exclusion of common causes fordiarrhea in infancy, followed by early endoscopy for tissuediagnosis, focusing on the crypt to villus ratio as a simplefirst-line diagnostic tool. Specific immunohistochemistrystaining in addition to the traditional H&E analysis furtherenhances the diagnostic process. The new algorithm alsoemploys early genetic testing, highlighting the fact that thevast majority of the CODEs with sustained symptoms aremonogenic disorders. Faster and accurate diagnosis ofneonatal and infantile diarrheas should improve patientcare, shorten length of stay, provide better prognosticationto children and their families, and will provide clinical andpathological information for improved genotype andphenotype characterization and association.

Many of the epithelial-specific CODE disorders stillrequire either life-long PN or allogenic intestinal

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transplantation.82 Transplanted patients require life-longhigh-dose immunosuppression that is associated with sig-nificant risk for acute and chronic opportunistic infections,allograft rejection/loss, and subsequent malignancy. Cell-based therapies of CRISPR/Cas9-corrected intestinal stemcells and autologous intestinal epithelial transplantationmay be a viable option in the coming years, but will requirenumerous advancements in several areas, including devel-oping ablation, implantation, and engraftment protocols.83

Further research via multicenter collaborations is essen-tial for both understanding the pathogenesis of CODE disor-ders and for developing new therapies targeted at these rarebut severe diseases. Investigation of CODEs will likelyenhance our understanding of the basic biology of the intes-tine greatly, and may provide insights into the pathogenesisand treatment of more common gastrointestinal diseases.

Supplementary MaterialNote: To access the supplementary material accompanyingthis article, visit the online version of Gastroenterology atwww.gastrojournal.org, and at https://doi.org/10.1053/j.gastro.2018.03.067.

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85.Höglund P, Sormaala M, Haila S, et al. Identification ofseven novel mutations including the first two genomicrearrangements in SLC26A3 mutated in congenitalchloride diarrhea. Hum Mutat 2001;18:233–242.

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Received October 5, 2017. Accepted March 17, 2018.

Reprint requestsAddress requests for reprints to: Yaron Avitzur, MD, Division ofGastroenterology, Hepatology and Nutrition, Department of Paediatrics, TheHospital for Sick Children, University of Toronto, 555 University Avenue,Toronto, Ontario, Canada M5G 1X8. e-mail: yaron.avitzur@sickkids.ca; fax:(416) 813-4972; or Martín G. Martín, MD, MPP, Department of Pediatrics,Division of Gastroenterology and Nutrition, Mattel Children’s Hospital andthe David Geffen School of Medicine, University of California Los Angeles,Los Angeles, California 90095. e-mail: MMartin@mednet.ucla.edu; fax: (310)206-0203.

Conflicts of interestThe authors disclose no conflicts.

Disorders of EpithelialNutrient/Electrolyte TransportCongenital Chloride Diarrhea

Congenital chloride diarrhea is characterized by achronic electrolyte transport–related diarrhea that starts inutero, commonly resulting in polyhydramnios in pregnancyand severe diarrhea at birth with concomitant hypo-chloremic hypokalemic metabolic alkalosis. A pathogno-monic feature of the diarrhea is a high fecal chloride level(>90 mmol/L) that can be used as an initial diagnostictest.53 Congenital chloride diarrhea is caused by mutationsin the Cl–/HCO3

– exchanger DRA (SLC26A3), which isrequired for electroneutral sodium absorption (in conjunc-tion with Naþ/Hþ exchange) particularly in the ileum andcolon. SLC26A3 mutations, therefore, result in a loss of in-testinal sodium-driven fluid absorption, with resultingprofuse watery diarrhea. More than 50 different mutationshave been reported with founder mutations in specificpopulations (eg, Finnish and Arab).84,85 Management ofcongenital chloride diarrhea revolves around preventingdehydration and alkalosis through enteral fluid and elec-trolyte supplementation. Oral administration of butyratewas found to reduce diarrheal output, although subsequentstudies have reported more equivocal results.86,87

Congenital Sodium DiarrheaCongenital sodium diarrhea (CSD) is characterized by a

severe electrolyte transport–related diarrhea that starts inutero, and infants are often born with dilated fluid-filledloops of intestine. In rare cases, infants can developpseudo-obstruction–like features and volvulus due todilated fluid-filled bowel. The diarrhea in CSD is character-ized by a high fecal Naþ content, and induces a metabolicacidosis. The genetic basis of CSD is heterogeneous,currently with 3 major known genes involved that primarilyimpact Naþ absorption in the intestine. Classical or non-syndromic CSD results from loss-of-function mutations inthe Na/Hþ exchanger NHE3, which is critical for normal Naþ

and fluid absorption from the intestine, as well as normalacid–base homeostasis.37 CSD also results from activatingmutations in the guanylin receptor GC-C (GUCY2C), resultingin elevated cellular cyclic guanosine monophosphate levels,inhibition of NHE3 function, and stimulation of chloridesecretion via CFTR chloride channels.50 Lastly, a syndromicform of CSD (SPINT2) that overlaps in phenotype with CTEhas been decribed.88 Infants with CSD typically requireintensive fluid, nutrient, and electrolyte support via PNduring the first year of life. Classical CSD caused by NHE3 orGUCY2C mutation appears to improve over time, witheventual weaning of PN. However, both NHE3 or GUCY2Cvariants appear to be associated with an increased risk ofIBD in later life.89

Glucose-Galactose MalabsorptionGlucose and galactose are 2 monosaccharides trans-

ported across the apical membrane by the Na-dependentglucose/galactose cotransporter (SLC5A1). Bi-allelic

loss-of-function mutations of SLC5A1 are associated with aselective form of malabsorption, glucose-galactose malab-sorption, which has been described thoroughly.49 While themajority of the missense mutations impaired the traffickingof the co-transporter to the plasma membrane, those thatdid reach the membrane provided important insight into thestructure of the transporter. Nonetheless, these childrenpresent with severe dehydration with a metabolic acidosis,and a diarrhea that ceases on a glucose/galactose-free diet.The dietary carbohydrate is limited to fructose lifelong,which is transported across the brush border by GLUT5(SLC2A5).

Primary Bile Acid DiarrheaAmong the rarest causes of CODE is a selective inability to

reabsorb bile salts in the intestinal portion of the enter-ohepatic circulation. A bi-allelic loss-of-function mutation ofthe sodium coupled bile salt re-uptake (SLC10A2) has beenassociated with watery diarrhea that stimulates colonic chlo-ride secretion and can be managed using a bile acid seques-trant.51 More recently bi-allelic loss-of-function mutations inthe basolateral bile acid transporter OST-b (SLC51B) havebeen associated with diarrhea, severe fat-soluble vitamindeficiency, and features of cholestatic liver disease.52

In contrast to the diarrhea that is associated withexcessive luminal bile acids, their deficiency is also associ-ated with diarrhea and malabsorption of fat. Certainly,inherited and non-inherited conditions associated withcholestatic liver disease and surgical resection of the distalbowel may result in steatorrhea, and as many as 6 rareselective autosomal recessive congenital defects of bile acidsynthesis have been described.98

Disorders of Epithelial Enzymesand MetabolismPrimary Lactase Deficiency

Lactose intolerance or hypolactasia is likely the mostcommon inherited disorder that results in a gastrointestinalphenotype. Hypolactasia is an autosomal recessive age-dependent mutation of the LPH gene that results inlactose-induced diarrhea after 5 years of age.54,55 Indeed,lactase non-persistence results from 2 variants located farupstream of the LPH gene that impairs enhancer activity.90

In contrast, primary lactase deficiency is exceedingly rare,and results from bi-allelic loss-of-function mutations of theLPH gene and leads to congenital diarrhea.56 It should benoted that transient lactose intolerance is common in pre-mature infants and older infants after acute gastroenteritis.Patients are managed on a lactose-free diet, and can besupplemented with oral lactase before the meal.

Sucrase-Isomaltase DeficiencyThe sucrase-isomaltase gene (SI) encodes 2 subunits,

sucrase and isomaltase, that form a heterodimer. Sucrasehydrolyzes sucrose, while isomaltase processes starch, iso-maltose, and maltose. Bi-allelic mutations of SI result in aloss of either sucrase, isomaltase, or both enzyme activities,

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and will result in diarrhea while on such a diet.57 Missensemutations have been shown to allow for either proper orimproper targeting to the plasma membrane, with the latterresulting in a combined sucrase and isomaltase deficiency.

Diacylglyceroltransferase-1A recently described CODE characterized by an electro-

lyte transport–related diarrhea, emesis, protein-losing en-teropathy (PLE), and growth failure induced by enteralintake of lipids was found to be due to a loss of functionmutation in DGAT1, which is involved in cellular triglycerideformation.32 More recently, hypomorphic mutations with aless severe phenotype have been reported.91 Managementof DGAT1 diarrhea relies on limiting enteral fat intake inconjunction with intravenous lipid administration to pre-vent essential fatty acid deficiency. The underlying mecha-nisms of how loss of DGAT1 function leads to the clinicalphenotype remain to be elucidated.

Disorders of Epithelial Traffickingand PolarityMicrovillus Inclusion Disease

Patients with MVID present with severe watery, elec-trolyte transport–related diarrhea and dehydration withfailure to thrive beginning in the first days or weeks of life.59

MVID results from inactivating mutations in myosin-Vb(MYO5B), a cytoskeletal motor protein, which regulatesapical membrane recycling through interaction with Rabsmall GTPases.33,58 MVID patients display pathognomonicmicrovillus inclusions by transmission electron microscopyin 10% of duodenal enterocytes.60 Other pathological hall-marks of the disease include villus atrophy, abnormal sub-apical periodic acid–Schiff/CD10 staining in enterocytes,inclusion staining with antibodies against ezrin or villin, anddiminution of brush-border microvillar markers, includingezrin, villin, phospho-ERM, NHE3, and DPP-IV.92–94

Abnormal transmission electron microscopy findings andimmunostaining suggest that the disease relates to impair-ments in Rab11A and Rab8A-dependent recycling andtrafficking of critical transporters to the apical and poten-tially basolateral membrane in the small bowel.92,95 Liverfailure associated with MVID patients may be related to theeffects of chronic PN. More recently a non-PN–relatedphenotype with normal GGT cholestasis was described inpatients demonstrating MYO5B mutations with and withoutthe MVID intestinal phenotype.63,96,97 Patients with MVIDrequire PN support for life and can benefit from liver/in-testine transplantation if they develop PN-associatedcomplications.

Other Microvillar EnteropathiesA truncation mutation of STX3 and mutation in STXBP2

elicit milder chronic diarrhea phenotypes.98,99 Patients withSTXBP2 mutations also display hemophagocytic lympho-histiyocytosis (HLH) type 5, requiring bone marrow trans-plantation (BMT) to treat their HLH. However, BMT has noeffect on the intestinal phenotype and PN is still required

post-transplantation. Both of these mutations show shortapical microvilli in enterocytes with the presence ofmicrovilli on the lateral membrane surfaces. This phenotyperesembles the pattern noted in mice with intestinally tar-geted deletion of Rab11a.100

Congenital Tufting EnteropathyChildren with CTE present with watery, sodium-losing

diarrhea in the first weeks of life. Mutations in the EPCAMgene are the cause for the majority of CTE cases and areassociated with the non-syndromic form of CTE that usuallyleads to isolated diarrhea.31,62 EPCAM is localized at thebasolateral membrane of the epithelial cell and plays a rolein the regulation of cell adhesion and proliferation viaClaudin-7.101 The syndromic form of CTE is characterized bychoanal atresia, rarely intestinal atresia, and chronic diar-rhea.88 Mutations of the SPINT2 gene that encodes a Kunitz-type protease inhibitor have been described in syndromicCTE, as well as in cases of congenital sodium diarrhea,probably representing phenotypic diversity associated withthe same mutation.102 The pathological hallmark of CTE isfocal epithelial tufts in the small and large bowel with villusatrophy. EPCAM staining is negative in cases of EPCAMmutation. Most CTE patients remain PN-dependent, butsome have improved over time and can tolerate variousamounts of enteral nutrition.

TTC7A DeficiencyTTC7A deficiency was recently described in children with

early-onset severe abnormal electrolyte transport diarrheaand enterocolitis.44 TTC7A is a TPR domain protein impor-tant in cell polarization and TTC7A deficiency leads to loss ofthe apicobasal polarity of the enterocyte, crypt–baseapoptosis, crypt and villus atrophy, and chronic inflamma-tion. The phenotype of TTC7A deficiency is diverse andranges from severe enterocolitis to multiple intestinalatresia with recurrence of the atresia after surgical resection.Severe combined immunodeficiency with T- and B-celllymphopenia and diminished function and hypo-gammaglobulinemia are integral parts of the disease.103–105

The prognosis is poor and almost all patients succumbduring infancy. The role of BMT in this population is ques-tionable, with limited improvement post-BMT and no effecton enterocyte structure and function.

Tricho-Hepato-Enteric SyndromeTricho-hepatic-enteric syndrome, also known as syn-

dromic diarrhea, is a multisystemic disease with abnormalintestinal function and hair and facial dysmorphism in allpatients, as well as liver disease in some. Woolly hair withtrichorhexis nodosa on hair analysis supports the diagnosis.intrauterine growth restriction, abnormal T-cell functionand antibody production, short stature, developmentaldelay, abnormal platelet morphology, and cardiac defectsare additional features of the disease. Small bowel biopsydemonstrates normal or mild to severe villus atrophyand variable infiltration of the lamina propria with mono-nuclear cells. Gastritis or colitis can appear in some cases.

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Tricho-hepatic-enteric syndrome is caused by mutations inthe TTC37 gene in 60% of the cases, while the remainder areassociated with SKIV2L mutation.106,107 Both gene productsform parts of the human Ski complex that have an antiviralrole. Prognosis is not only influenced by PN use andmalabsorption, but also by the increased risk of infectionsand progressive liver disease.

PLVAP DeficiencyMutations in the plasmalemma vesicle–associated pro-

tein (PLVAP) were recently found to result in a very severeform of PLE characterized by hypoproteinemia, hypo-albuminemia, and hypertriglyceridemia. Loss of PLVAPexpression results in deletion of the diaphragms of endo-thelial fenestrae, leading to plasma protein extravasationand PLE, with early fatality at 5 months of age in the onlycase reported so far.36

Disorders of EnteroendocrineCell FunctionEnteric Anendocrinosis (NEUROG3)

The original disorder in this group, enteric anendocri-nosis, results from bi-allelic loss-of-function mutations ofNeurogenin3, a basic helix–loop–helix transcription factor,which, in mice, is required for enteroendocrine and b-celldevelopment.64,65 Intestinal biopsies reveal a normal cryptto villus ratio, and a selective loss of all types of EECs. Thesechildren eventually develop an insulin-dependent diabetesbeyond the third year of life, with an absence of autoanti-bodies. The lack of hyperglycemia during early infancy wasoriginally attributed to a hypomorph missense mutation.However, subsequent cases with severe nonsense mutationsand an absence of diabetes suggest that, unlike mice, othertranscription factors may be sufficient to generate a b-cellmass that is sufficient to maintain normoglycemia intochildhood in the absence of Neurogenin3. Recent studiesindicate that mature enteroendocrine cells function as atleast 1 of the quiescent stem cell populations that can de-differentiate to active stem cells after injury, suggestingthat children with this disorder may have a limited reservecapacity to replenish stem cells after stem cell injury.108

Enteric Dysendocrinosis (PCSK1)Enteric dysendocrinosis is an autosomal recessive dis-

order resulting from loss-of-function mutations of PCSK1,coding for prohormone convertase (PC1/3), a protease thatis required for the biosynthetic processing of hormoneprecursors into their fully functional forms.66 The earlyclinical features mimic those of its anendocrinosis coun-terpart with generalized malabsorption that will require PNat least for the first several years, despite normal-appearingcrypt–villus units. A distinguishing feature of children withimpaired PC1/3 function is the early development of abroad group of systemic endocrinopathies, including adre-nal insufficiency, hypothyroidism, and diabetes insipidus,among others; although, diabetes mellitus is not among

them.109 PC1/3 also processes neuropeptides in the hypo-thalamus that are required for energy homeostasis andcontrol of appetite, including pro-opiomelanocortin, andexplains the biphasic nature of this condition. During earlyinfancy, these children are malnourished, have severediarrhea, and require PN, but subsequently develop a pro-found polyphagia and modest obesity, despite ongoingdiarrhea. Pro-insulin is markedly elevated in these patientsand can serve as a diagnostic test.

Other Endocrinopathies (RFX6/ARX)Mitchell-Riley syndrome is a complex clinical condition

associated with intestinal atresia, malrotation, intrinsic andextrinsic biliary duct abnormalities, and typically neonataldiabetes among many others abnormalities.67 This disorderresults from a bi-allelic mutation of RFX6, a transcriptionfactor that functions both up- and downstream of NEUROG3.These children have a malabsorptive diarrhea that was onceattributed to the complex gastrointestinal malformations,but it mimics other enteric endocrinopathies and biopsiesreveal EECs. Recent data suggest that RFX6 may also berequired for production of components of the insulinsecretion pathway and is therefore required for b-cellfunction, and may contribute to the early age at onset ofdiabetes.110

ARX is an X-linked gene that encodes a homeobox tran-scription factor whose loss-of-function mutations result in acomplex of clinical features of lissencephaly (smoothcerebral cortex), seizure disorder, ambiguous genitalia,and malabsorptive diarrhea. Limited studies in mice andhumans suggest a possible selective reduction of GLP-1 andcholecystokinin-expressing EECs.68,69

Immune Dysregulation–AssociatedEnteropathiesFOXP3

Mutations in the FOXP3 gene are known to cause IPEXsyndrome.70,71 The gene is located on X chromosome and,therefore, males are affected. Typically, these patients pre-sent before 6 months of age with severe watery diarrheathat does not respond to nil per os, and PN is required.111

The intestinal pathological features vary from completevillus atrophy with apoptosis in a graft-vs-host appearance,to loss of goblet and Paneth cells, and mild inflammation.FOXP3 mutations should be considered in all male patientswith persistent diarrhea, especially those with diabetes,severe eczema, and/or thyroiditis. Patients ultimatelyrequire BMT, but may be managed with sirolimus thatattenuates effector T cells.112

X-Linked Inhibitor of ApoptosisX-linked inhibitor of apoptosis is encoded by the BIRC4

gene, initially described in patients with X-linked HLH syn-drome.113 However, it is now known that X-linked inhibitorof apoptosis patients may present with intestinal diseasethat varies from villous atrophy to severe enterocolitis with

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perianal disease.114 Unlike other CODE disorders, the onsetof symptoms occurs from less than 6 months of age to up to40 years of age. A high degree of suspicion is needed forpatients with diarrhea and biochemical features of HLH.

LRBA/CTLA4Recently, a new multisystemic disease with autoimmu-

nity, primary immunodeficiency, and autoimmune enterop-athy as the main feature was described and caused by 2distinct genes. CTLA4 mutations were identified as auto-somal dominant with variable penetrance,115 while LRBA116

mutations cause a phenocopy due to autosomal recessivemutations. CTLA4 functions as an early checkpoint con-trolling T-cell response to antigens. LRBA is expressed onRab11-positive recycling endosomes and appears to func-tion in CTLA4 recycling.117 Patients with CTLA4 and LRBAmutations have a variable age of presentation from the firstdays of life to adulthood. They may have severe villus at-rophy with IPEX-like autoimmune features, including auto-immune enteropathy and common variable immunedeficiency features. These patients may be treated withabatacept, a drug containing the extracellular domain ofCTLA4. BMT has been shown to be a potential long-termtherapy.

CD55 Deficiency (CHAPLE Syndrome)CD55 deficiency with hyperactivation of complement,

angiopathic thrombosis, and PLE (CHAPLE syndrome).118

Most patients present in the first 2 years of life with PLEdue to primary intestinal lymphangiectasia, abdominal pain,thromboembolic disease, recurrent infections, and variousdegrees of bowel inflammation. Mutations in the geneencoding CD55, a complement regulator, lead to hyper-activation of complement. Treatment with eculizumab, acomplement inhibitor, leads to improvement in PLEsymptoms.

ADAM17 DeficiencyADAM17 is a protein with metalloprotease properties

that cleaves and activates numerous membrane-boundprecursors, including TNFa, EGF, and Notch. Bi-allelic mu-tations of ADAM17 have been described in several patientswith a severe skin rash; bloody/watery diarrhea; abnormalhair and disorganized eyelashes and eyebrows; and recur-rent skin, nail, and enteric infections.119,120 The diseaseappears in the first days of life, and 2 of the 3 patientsdescribed died during childhood. Small bowel biopsies showvillus blunting, lengthening of crypts, and mononuclear cellinfiltrates.

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Supplementary Figure 1. Functional testing of CODEs. (A) Multiplex immunocytochemistry showing 40,6-diamidino-2-phenylindole (DAPI), NaþKþ ATPase, pan cadherin, segmentation analysis of cell boundaries, virtual H&E section derivedfrom staining and multicolor overlay of the same colon tissue sections. (B) Swelling assay in human intestinal enteroids.Example shows control human enteroids isolated after 5 days in culture and placed in custom microfluidic device allowingfor capture of enteroids.121 Enteroids (arrow) are stimulated with 50 mM forskolin and swelling followed over time (minutes)indicating robust fluid secretion. (C) Example of cell trafficking assay (transcytosis) using IgG transport via the epithelial FcRnreceptor as a method of measuring trafficking pathways. Polarized epithelial cells (MDCK) expressing FcRN receptor aregrown on permeable supports. FcRn-dependent IgG trafficking in monolayers depleted of the indicated genes (red) iscompared with IgG trafficking in control monolayers transfected with nontargeting endoribonuclease-prepared small inter-fering RNA (esiRNA) (black). Transcytosis is normalized to the 90-minute time point of matched control monolayers transfectedwith nontargeting esiRNA, and internalization experiments were normalized to the matched nontargeting control. Eachexperiment was conducted in triplicate on 2–4 independent days; the number of replicates and days are indicated below eachbar graph in the form of n ¼ (replicates per experiment) � (number of independent experiments). *P < .05; as estimated by theWilcoxon signed-rank test, ***P < .0001. Plots show the weighted mean ± SD across experiments. Right: examples of inhi-bition of transcytosis showing time course of apically directed IgG transcytosis as assessed by 60-minute pulse and 90-minutechase after knockdown of key trafficking genes (exocyst complex components 2 and 7).81

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Supplementary Table 1.Antibodies for Immunohistochemistry Staining to Assess CODE

Antibody/staina Cells/structure Disorder or cell-specific

CD10 Enterocytes–brush border MVIDPeriodic–acid Schiff Goblet cells–enterocyte brush border MVID, DGAT1, AIEVillin Enterocytes–brush border MVIDCarcinoembryonic antigen (CEA, CEACAM5) Enterocytes–brush border MVIDAlkaline phosphatase Enterocytes–brush border MVIDF-actin Enterocytes–brush border MVIDEPCAM (clone: MOC31) Enterocytes–basolateral cell membrane CTEOil Red O Enterocytes–intracellular lipid ABL, HBL, CRDClaudin-7 Enterocytes–lateral junctions CTE, Synd CSDLysozyme Paneth cells–granules AIEChromogranin A Enteroendocrine cells’ neuroendocrine granules Enteric anendocrinosisSynaptophysin Enteroendocrine cells’ neuroendocrine granules Enteric anendocrinosisPC-1 Enteroendocrine cells PC1/3 deficiencyGP2 Microfold (M) cells Cell-specificPTGS1 Tuft cells Cell-specificKi67, PCNA Proliferating cells Cell-specificPhospho-histone H3 Proliferating cells Cell-specificCD20 B cells Cell-specificCD3/CD4/CD8 T cells (CD3)

T-helper cells (CD4)Cytotoxic T cells (CD8)

Cell-specific

PU.1/CD68 Macrophages Cell-specificCD138 Plasma cells Cell-specificActivated caspase-3 Cell apoptosis Cell-specific

AIE, autoimmune enteropathy; CSD, congenital sodium diarrhea; PCNA, proliferating cell nuclear antigen.aHistochemical stains in italics; remaining stains are immunohistochemistry.

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