CODE Genes

Disorders Of Epithelial Nutrient/Electrolyte Transport

Congenital Chloride Diarrhea

Congenital chloride diarrhea is characterized by a chronic electrolyte transport-related diarrhea that starts in utero, commonly resulting in polyhydramnios in pregnancy and severe diarrhea at birth with concomitant hypochloremic hypokalemic metabolic alkalosis. A pathognomonic feature of the diarrhea is a high fecal chloride level (>90 mmol/L) that can be used as an initial diagnostic test.53 Congenital chloride diarrhea is caused by mutations in the Cl/HCO3 exchanger DRA (SLC26A3), which is required for electroneutral sodium absorption (in conjunction with Na+/H+ exchange) particularly in the ileum and colon. SLC26A3 mutations, therefore, result in a loss of intestinal sodium-driven fluid absorption, with resulting profuse watery diarrhea. More than 50 different mutations have been reported with founder mutations in specific populations (eg, Finnish and Arab).84,85 Management of congenital chloride diarrhea revolves around preventing dehydration and alkalosis through enteral fluid and electrolyte supplementation. Oral administration of butyrate was found to reduce diarrheal output, although subsequent studies have reported more equivocal results.86,87

Congenital Sodium Diarrhea

Congenital sodium diarrhea (CSD) is characterized by a severe electrolyte transport-related diarrhea that starts in utero, and infants are often born with dilated fluid-filled loops of intestine. In rare cases, infants can develop pseudo-obstruction-like features and volvulus due to dilated fluid-filled bowel. The diarrhea in CSD is characterized by a high fecal Na+ content, and induces a metabolic acidosis. The genetic basis of CSD is heterogeneous, currently with 3 major known genes involved that primarily impact Na+ absorption in the intestine. Classical or non-syndromic CSD results from loss-of-function mutations in the Na/H+ exchanger NHE3, which is critical for normal Na+ and fluid absorption from the intestine, as well as normal acid-base homeostasis.37 CSD also results from activating mutations in the guanylin receptor GC-C (GUCY2C), resulting in elevated cellular cyclic guanosine monophosphate levels, inhibition of NHE3 function, and stimulation of chloride secretion via CFTR chloride channels.50 Lastly, a syndromic form of CSD (SPINT2) that overlaps in phenotype with CTE has been decribed.88 Infants with CSD typically require intensive fluid, nutrient, and electrolyte support via PN during the first year of life. Classical CSD caused by NHE3 or GUCY2C mutation appears to improve over time, with eventual weaning of PN. However, both NHE3 or GUCY2C variants appear to be associated with an increased risk of IBD in later life.89

Glucose-Galactose Malabsorption

Glucose and galactose are 2 monosaccharides transported across the apical membrane by the Na-dependent glucose/galactose cotransporter (SLC5A1). Bi-allelic loss-of-function mutations of SLC5A1 are associated with a selective form of malabsorption, glucose-galactose malabsorption, which has been described thoroughly.49 While the majority of the missense mutations impaired the trafficking of the co-transporter to the plasma membrane, those that did reach the membrane provided important insight into the structure of the transporter. Nonetheless, these children present 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 Diarrhea

Among 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 of the sodium coupled bile salt re-uptake (SLC10A2) has been associated with watery diarrhea that stimulates colonic chloride secretion and can be managed using a bile acid seques-trant.51 More recently bi-allelic loss-of-function mutations in the basolateral bile acid transporter OST-β (SLC51B) have been associated with diarrhea, severe fat-soluble vitamin deficiency, and features of cholestatic liver disease.52

In contrast to the diarrhea that is associated with excessive luminal bile acids, their deficiency is also associated with diarrhea and malabsorption of fat. Certainly, inherited and non-inherited conditions associated with cholestatic liver disease and surgical resection of the distal bowel may result in steatorrhea, and as many as 6 rare selective autosomal recessive congenital defects of bile acid synthesis have been described.98

Disorders Of Epithelial Enzymes And Metabolism

Primary Lactase Deficiency

Lactose intolerance or hypolactasia is likely the most common inherited disorder that results in a gastrointestinal phenotype. Hypolactasia is an autosomal recessive age- dependent mutation of the LPH gene that results in lactose-induced diarrhea after 5 years of age.54,55 Indeed, lactase non-persistence results from 2 variants located far upstream 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 the LPH gene and leads to congenital diarrhea.56 It should be noted that transient lactose intolerance is common in premature infants and older infants after acute gastroenteritis. Patients are managed on a lactose-free diet, and can be supplemented with oral lactase before the meal.

Sucrase-Isomaltase Deficiency

The sucrase-isomaltase gene (SI) encodes 2 subunits, sucrase and isomaltase, that form a heterodimer. Sucrase hydrolyzes sucrose, while isomaltase processes starch, isomaltose, and maltose. Bi-allelic mutations of SI result in a loss of either sucrase, isomaltase, or both enzyme activities, and will result in diarrhea while on such a diet.57 Missense mutations have been shown to allow for either proper or improper targeting to the plasma membrane, with the latter resulting in a combined sucrase and isomaltase deficiency.

Diacylglyceroltransferase-1

A recently described CODE characterized by an electrolyte transport-related diarrhea, emesis, protein-losing enteropathy (PLE), and growth failure induced by enteral intake of lipids was found to be due to a loss of function mutation in DGAT1, which is involved in cellular triglyceride formation.32 More recently, hypomorphic mutations with a less severe phenotype have been reported.91 Management of DGAT1 diarrhea relies on limiting enteral fat intake in conjunction with intravenous lipid administration to prevent essential fatty acid deficiency. The underlying mechanisms of how loss of DGAT1 function leads to the clinical phenotype remain to be elucidated.

Disorders Of Epithelial Trafficking And Polarity

Microvillus Inclusion Disease

Patients with MVID present with severe watery, electrolyte transport-related diarrhea and dehydration with failure 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 regulates apical membrane recycling through interaction with Rab small GTPases.33,58 MVID patients display pathognomonic microvillus inclusions by transmission electron microscopy in 10% of duodenal enterocytes.60 Other pathological hallmarks of the disease include villus atrophy, abnormal subapical periodic acid-Schiff/CD10 staining in enterocytes, inclusion staining with antibodies against ezrin or villin, and diminution of brush-border microvillar markers, including ezrin, villin, phospho-ERM, NHE3, and DPP-IV.9294 Abnormal transmission electron microscopy findings and immunostaining suggest that the disease relates to impairments in Rab11A and Rab8A-dependent recycling and trafficking of critical transporters to the apical and potentially basolateral membrane in the small bowel.92,95 Liver failure associated with MVID patients may be related to the effects of chronic PN. More recently a non-PN-related phenotype with normal GGT cholestasis was described in patients demonstrating MYO5B mutations with and without the MVID intestinal phenotype.63,96,97 Patients with MVID require PN support for life and can benefit from liver/in-testine transplantation if they develop PN-associated complications.

Other Microvillar Enteropathies

A truncation mutation of STX3 and mutation in STXBP2 elicit milder chronic diarrhea phenotypes.98,99 Patients with STXBP2 mutations also display hemophagocytic lympho-histiyocytosis (HLH) type 5, requiring bone marrow transplantation (BMT) to treat their HLH. However, BMT has no effect on the intestinal phenotype and PN is still required post-transplantation. Both of these mutations show short apical microvilli in enterocytes with the presence of microvilli on the lateral membrane surfaces. This phenotype resembles the pattern noted in mice with intestinally targeted deletion of Rab11a.100

Congenital Tufting Enteropathy

Children with CTE present with watery, sodium-losing diarrhea in the first weeks of life. Mutations in the EPCAM gene are the cause for the majority of CTE cases and are associated with the non-syndromic form of CTE that usually leads to isolated diarrhea.31,62 EPCAM is localized at the basolateral membrane of the epithelial cell and plays a role in the regulation of cell adhesion and proliferation via Claudin-7.101 The syndromic form of CTE is characterized by choanal atresia, rarely intestinal atresia, and chronic diar-rhea.88 Mutations of the SPINT2 gene that encodes a Kunitztype protease inhibitor have been described in syndromic CTE, as well as in cases of congenital sodium diarrhea, probably representing phenotypic diversity associated with the same mutation.102 The pathological hallmark of CTE is focal epithelial tufts in the small and large bowel with villus atrophy. EPCAM staining is negative in cases of EPCAM mutation. Most CTE patients remain PN-dependent, but some have improved over time and can tolerate various amounts of enteral nutrition.

TTC7A Deficiency

TTC7A deficiency was recently described in children with early-onset severe abnormal electrolyte transport diarrhea and enterocolitis.44 TTC7A is a TPR domain protein important in cell polarization and TTC7A deficiency leads to loss of the apicobasal polarity of the enterocyte, crypt-base apoptosis, crypt and villus atrophy, and chronic inflammation. The phenotype of TTC7A deficiency is diverse and ranges from severe enterocolitis to multiple intestinal atresia with recurrence of the atresia after surgical resection. Severe combined immunodeficiency with T- and B-cell lymphopenia and diminished function and hypo-gammaglobulinemia are integral parts of the disease. The prognosis is poor and almost all patients succumb during infancy. The role of BMT in this population is questionable, with limited improvement post-BMT and no effect on enterocyte structure and function.

Tricho-Hepato-Enteric Syndrome

Tricho-hepatic-enteric syndrome, also known as syndromic diarrhea, is a multisystemic disease with abnormal intestinal function and hair and facial dysmorphism in all patients, as well as liver disease in some. Woolly hair with trichorhexis nodosa on hair analysis supports the diagnosis. intrauterine growth restriction, abnormal T-cell function and antibody production, short stature, developmental delay, abnormal platelet morphology, and cardiac defects are additional features of the disease. Small bowel biopsy demonstrates normal or mild to severe villus atrophy and variable infiltration of the lamina propria with mononuclear cells. Gastritis or colitis can appear in some cases. Tricho-hepatic-enteric syndrome is caused by mutations in the TTC37 gene in 60% of the cases, while the remainder are associated with SKIV2L mutation.106,107 Both gene products form parts of the human Ski complex that have an antiviral role. Prognosis is not only influenced by PN use and malabsorption, but also by the increased risk of infections and progressive liver disease.

PLVAP Deficiency

Mutations in the plasmalemma vesicle-associated protein (PLVAP) were recently found to result in a very severe form of PLE characterized by hypoproteinemia, hypo-albuminemia, and hypertriglyceridemia. Loss of PLVAP expression results in deletion of the diaphragms of endothelial fenestrae, leading to plasma protein extravasation and PLE, with early fatality at 5 months of age in the only case reported so far.36

Disorders Of Enteroendocrine Cell Function

Enteric Anendocrinosis (NEUROG3)

The original disorder in this group, enteric anendocri-nosis, results from bi-allelic loss-of-function mutations of Neurogenin3, a basic helix-loop-helix transcription factor, which, in mice, is required for enteroendocrine and β-cell development.64,65 Intestinal biopsies reveal a normal crypt to villus ratio, and a selective loss of all types of EECs. These children eventually develop an insulin-dependent diabetes beyond the third year of life, with an absence of autoantibodies. The lack of hyperglycemia during early infancy was originally attributed to a hypomorph missense mutation. However, subsequent cases with severe nonsense mutations and an absence of diabetes suggest that, unlike mice, other transcription factors may be sufficient to generate a b-cell mass that is sufficient to maintain normoglycemia into childhood in the absence of Neurogenin3. Recent studies indicate that mature enteroendocrine cells function as at least 1 of the quiescent stem cell populations that can dedifferentiate to active stem cells after injury, suggesting that children with this disorder may have a limited reserve capacity to replenish stem cells after stem cell injury.108

Enteric Dysendocrinosis (PCSK1)

Enteric dysendocrinosis is an autosomal recessive disorder resulting from loss-of-function mutations of PCSK1, coding for prohormone convertase (PC1/3), a protease that is required for the biosynthetic processing of hormone precursors into their fully functional forms.66 The early clinical features mimic those of its anendocrinosis counterpart with generalized malabsorption that will require PN at least for the first several years, despite normal-appearing crypt-villus units. A distinguishing feature of children with impaired PC1/3 function is the early development of a broad group of systemic endocrinopathies, including adrenal insufficiency, hypothyroidism, and diabetes insipidus, among others; although, diabetes mellitus is not among them.109 PC1/3 also processes neuropeptides in the hypothalamus that are required for energy homeostasis and control of appetite, including pro-opiomelanocortin, and explains the biphasic nature of this condition. During early infancy, these children are malnourished, have severe diarrhea, and require PN, but subsequently develop a profound polyphagia and modest obesity, despite ongoing diarrhea. Pro-insulin is markedly elevated in these patients and can serve as a diagnostic test.

Other Endocrinopathies (RFX6/ARX)

Mitchell-Riley syndrome is a complex clinical condition associated with intestinal atresia, malrotation, intrinsic and extrinsic biliary duct abnormalities, and typically neonatal diabetes among many others abnormalities.67 This disorder results from a bi-allelic mutation of RFX6, a transcription factor that functions both up- and downstream of NEUROG3. These children have a malabsorptive diarrhea that was once attributed to the complex gastrointestinal malformations, but it mimics other enteric endocrinopathies and biopsies reveal EECs. Recent data suggest that RFX6 may also be required for production of components of the insulin secretion pathway and is therefore required for β-cell function, and may contribute to the early age at onset of diabetes.110

ARX is an X-linked gene that encodes a homeobox transcription factor whose loss-of-function mutations result in a complex of clinical features of lissencephaly (smooth cerebral cortex), seizure disorder, ambiguous genitalia, and malabsorptive diarrhea. Limited studies in mice and humans suggest a possible selective reduction of GLP-1 and cholecystokinin-expressing EECs.68,69

Immune Dysregulation-Associated Enteropathies

FOXP3

Mutations in the FOXP3 gene are known to cause IPEX syndrome.70,71 The gene is located on X chromosome and, therefore, males are affected. Typically, these patients present before 6 months of age with severe watery diarrhea that does not respond to nil per os, and PN is required.111 The intestinal pathological features vary from complete villus 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 patients with persistent diarrhea, especially those with diabetes, severe eczema, and/or thyroiditis. Patients ultimately require BMT, but may be managed with sirolimus that attenuates effector T cells.112

X-Linked Inhibitor of Apoptosis

X-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 inhibitor of apoptosis patients may present with intestinal disease that varies from villous atrophy to severe enterocolitis with perianal disease.114 Unlike other CODE disorders, the onset of symptoms occurs from less than 6 months of age to up to 40 years of age. A high degree of suspicion is needed for patients with diarrhea and biochemical features of HLH.

LRBA/CTLA4

Recently, a new multisystemic disease with autoimmunity, primary immunodeficiency, and autoimmune enteropathy as the main feature was described and caused by 2 distinct genes. CTLA4 mutations were identified as autosomal dominant with variable penetrance,115 while LRBA116 mutations cause a phenocopy due to autosomal recessive mutations. CTLA4 functions as an early checkpoint controlling T-cell response to antigens. LRBA is expressed on Rab11-positive recycling endosomes and appears to function in CTLA4 recycling.117 Patients with CTLA4 and LRBA mutations have a variable age of presentation from the first days of life to adulthood. They may have severe villus atrophy with IPEX-like autoimmune features, including autoimmune enteropathy and common variable immune deficiency features. These patients may be treated with abatacept, a drug containing the extracellular domain of CTLA4. BMT has been shown to be a potential long-term therapy.

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 PLE due to primary intestinal lymphangiectasia, abdominal pain, thromboembolic disease, recurrent infections, and various degrees of bowel inflammation. Mutations in the gene encoding CD55, a complement regulator, lead to hyperactivation of complement. Treatment with eculizumab, a complement inhibitor, leads to improvement in PLE symptoms.

ADAM17 Deficiency

ADAM17 is a protein with metalloprotease properties that cleaves and activates numerous membrane-bound precursors, including TNFα, EGF, and Notch. Bi-allelic mutations of ADAM17 have been described in several patients with a severe skin rash; bloody/watery diarrhea; abnormal hair and disorganized eyelashes and eyebrows; and recurrent skin, nail, and enteric infections.119,120 The disease appears in the first days of life, and 2 of the 3 patients described died during childhood. Small bowel biopsies show villus blunting, lengthening of crypts, and mononuclear cell infiltrates.