Chronic intestinal pseudo‐obstruction in adults: A practical guide to identify patient subgroups that are suitable for more specific treatments

Chronic intestinal pseudo‐obstruction is a rare and heterogeneous syndrome characterized by recurrent symptoms of intestinal obstruction with radiological features of dilated small or large intestine with air/fluid levels in the absence of any mechanical occlusive lesion. Several diseases may be associated with chronic intestinal pseudo‐obstruction and in these cases, the prognosis and treatment are related to the underlying disease. Also, in its “primary or idiopathic” form, two subgroups of patients should be determined as they require a more specific therapeutic approach: patients whose chronic intestinal pseudo‐obstruction is due to sporadic autoimmune/inflammatory mechanisms and patients whose neuromuscular changes are genetically determined. In a context of a widely heterogeneous adult population presenting chronic intestinal pseudo‐obstruction, this review aims to summarize a practical diagnostic workup for identifying definite subgroups of patients who might benefit from more specific treatments, based on the etiology of their underlying condition.

0][11] CIPO is also different from acute intestinal pseudo-obstruction or Ogilvie syndrome, in which the neuromuscular architecture of the intestinal wall is preserved but a transient and reversible imbalance of excitatory and inhibitory neurotransmitters is responsible for acute dilation of the colon and/or small intestine. 17,18veral diseases (Table 1) are associated with CIPO, such as Hirschsprung's disease, mitochondrial neuro-gastro-intestinal encephalomyopathy (MNGIE), or paraneoplastic CIPO.In these conditions, treatment and prognosis are strongly linked with those of the underlying disease.In the remaining cases, CIPO seems "primary" or "idiopathic," but two subgroups can be recognized among them: those in whom CIPO is caused by a sporadic abnormal autoimmune/ inflammatory response directed against neural or muscular structures of the intestine, and those in whom an inherited or de novo genetic mutation underlies the impairment of muscle, neural, or interstitial cell function.The early recognition of these subgroups is clinically relevant, since those with autoimmune/inflammatory forms might benefit from immunosuppressive treatment 19 and those with congenital forms might benefit from a more precise definition of the underlying pathophysiology, 20,21 genetic counseling, and hopefully, in the future, gene therapy. 22e purpose of this review will be to summarize the diagnostic workup that can be applied to adult patients with CIPO, with a specific focus on identifying patients with autoimmune or genetic forms of the condition.

| THE D IAG NOS IS OF CIP O
The diagnosis of CIPO is difficult, as evidenced by the median interval of 8 years between symptom onset and diagnosis, 23 during which patients complain of a long history of recurrent episodes of vomiting and abdominal distention, variably accompanied by radiological signs of intestinal obstruction (dilatation of the bowel associated with air-fluid levels).
The first step in diagnosis is the careful exclusion of mechanical obstruction.Abdominal magnetic resonance imaging (MRI) or computed tomography (CT) scan is recommended at the onset of symptoms to exclude an intrinsic or extrinsic occlusive lesion.Imaging should be combined with endoscopic evaluation of the site of "pseudo-obstruction" caudal to the distended segment of the bowel, in order to rule out organic stenosis, congenital occlusive membrane, or atresia. 24Medications that impair gastrointestinal motility, such as opioids, tricyclic antidepressants, anticholinergics, ganglionic blockers, clonidine, and phenothiazines, should be discontinued, when possible, as they can exacerbate CIPO. 25 Symptoms and signs of bowel dilatation must have been present for at least 6 months to verify that the alteration underlying bowel dilatation is chronic. 6Persistence of bowel dilatation can be confirmed by MRI of the bowel, which is preferred over CT to minimize X-ray exposure in patients who will undergo repeated radiological investigations.A low-fiber diet and avoidance of fluid overload before the examination are recommended.Although dilatation of the bowel is a cornerstone for diagnosis, it should be recognized that there are no internationally accepted criteria for defining the normal diameter of different tracts of the bowel and that dilatation by itself does not provide specific clues to the underlying motor disorder.[16]

| D IS E A S E S A SSO CIATED WITH CIP O
Once the diagnosis of CIPO has been established, the next step is to consider whether the syndrome is associated to diseases with defined prognosis and treatment (Table 1 and Figure 1).A careful history and physical examination, including neurological evaluation, are essential to guide the recognition of these diseases and to limit the use of unnecessary diagnostic tests.This is important given the low probability that CIPO might be the onset presentation of these previously unrecognized diseases.Table 2 summarizes the tests and investigations that can be requested in this diagnostic process.
Hirschsprung's disease is characterized by the presence of megacolon and is diagnosed in <5% of patients in adulthood. 26,27The hypertonic segment begins in the cranial rectum and ends in the sigmoid-descending colon transition zone. 26At anorectal manometry, the physiological recto-anal inhibitory reflex induced by rectal distension, is absent. 28The diagnosis should be confirmed by the absence of ganglia and the abundance of cholinergic nerve fibers in the rectal biopsy performed at least 2 cm above the dentate line to avoid the physiologic aganglionic zone in the caudal rectum. 29Most cases of Hirschsprung's disease are sporadic and are associated with mutations in the receptor tyrosine kinase proto-oncogene. 30rschsprung's disease also often occurs in association with genetic syndromes, 31 such as Down syndrome 32,33 or multiple endocrine neoplasia 2B. 34GIE is characterized by the association of gastrointestinal dysmotility, peripheral neuropathy, chronic progressive external ophthalmoplegia, and leukoencephalopathy, which generally appear between the ages of 10 and 40 years of age.Gastrointestinal symptoms evolve from mild to severe and include dysphagia, abdominal pain, vomiting, diarrhea and weight loss. 35,36Creatinine phosphatase kinase (CPK), lactate dehydrogenase (LDH), and plasma lactate levels may be increased.Brain MRI reveals cerebral leukoencephalopathy. 36 MNGIE is an autosomal recessive syndrome caused by homozygous and compound heterozygous mutations in the TYMP gene, encoding the thymidine phosphorylase enzyme that regulates the mitochondrial nucleotide pool. 37Other mitochondrial DNA F I G U R E 1 Diagnostic flow chart in adult patients with chronic intestinal pseudo-obstruction (CIPO).Diseases associated with CIPO are reported in Table 1.Tests and investigations that can be applied during the diagnostic process are detailed in Table 2.
depletion syndromes characterized by progressive multisystem disorder presenting also with chronic gastrointestinal dysmotility and CIPO, such as mitochondrial DNA depletion syndrome-4A and -4B, are caused by compound heterozygous mutations in POLG, an autosomic gene encoding the mitochondrial DNA polymerase. 380][41] Diagnosis is confirmed by genetic testing for heterozygous pathogenic trinucleotide repeat expansion (CTG)n in the 3-prime untranslated region of the DMPK gene (myotonic dystrophy type 1) or tetranucleotide expansion in the CNBP gene (myotonic dystrophy type 2), or for disrupting mutations in the DMD gene, on the X chromosome, which encode dystrophin (Duchenne) synthesis. 42sautonomic symptoms (orthostatic hypotension, excess or inability to sweat, abnormally fast or slow heart rate after exercise, increased salt secretion, rhinorrhea, erectile problems) associated with altered tests of autonomic function 43 suggest pre-ganglionic or post-ganglionic lesions affecting both sympathetic and parasympathetic nerves. 44Anti-ganglionic acetylcholine receptor antibodies (anti-gAChR) may be increased. 45,46Autonomic dysfunction should suggest multisystem atrophy in patients with parkinsonism who are less responsive to levodopa, and in patients with cerebellar ataxia 47 or amyloidosis, since amyloid infiltrates both the intestinal mucosa and neuromuscular structures and extrinsic autonomic peripheral nerves.9][50] The familial visceral form of amyloidosis is a hereditary generalized amyloidosis in which viscera are particularly affected.It is caused by autosomal dominant mutations in the apolipoprotein A1 gene (APOA1), in the fibrinogen alpha-chain gene (FGA), or in the lysozyme gene (LYZ). 51PO has been reported in association with celiac disease 52 that should be screened with total immunoglobulin A, and tissue anti-transglutaminase antibodies.Anti-nuclear antibodies (ANA) can be used to screen for systemic lupus erythematosus. 53,54flammatory myositis of striated muscle (myositis, dermatomyositis) can present with CIPO.Inflammatory myositis is now classified under the anti-synthetase syndrome, based on the presence of eight types of antibodies directed against aminoacyl-tRNA synthetase, an enzyme that attaches the appropriate amino acid to the corresponding tRNA.These antibodies were increased in about 40% of patients with striated muscle myositis, but the presence of anti-theonyl-tRNA synthetase (Anti-PL-7) antibodies was also reported in two patients with CIPO 55 and the presence of anti-glycine-tRNA synthetase (Anti-EJ) antibodies was reported in a case of dermatomyositis presenting with proximal muscle weakness, myalgia, heliotropic rash, elevated CPK levels, and CIPO. 56stemic sclerosis is suggested by skin involvement, Raynaud's phenomenon, microvascular abnormalities on capillaroscopy, and the presence of anti-topoisomerase I (SCL 70) antibodies. 57,58 B L E 2 Tests and investigations according to the suspected diagnosis in adult patients presenting with chronic intestinal pseudoobstruction (CIPO).

Diagnosis Tests and investigations
Hirschsprung Several infections can cause CIPO, including Borrelia burgdorferi (Lyme disease), 59 Stongyloides stercoralis, 60 and Trypanosoma cruzi (Chagas disease). 61Laboratory investigations should be guided by the clinical history of exposure in endemic areas and may be screened by serum anti-Borrelia, anti-Strongyloides, and anti-Trypanosoma cruzi antibodies.
Advanced age of onset, the presence of neurological symptoms, and concomitant neoplasm (particularly small cell lung cancer) suggest paraneoplastic CIPO.Anti-Hu onconeural antibodies are often present in these cases. 62,63

| AUTOIMMUNE/INFL AMMATORY CIP O
CIPO may be the sporadic manifestation of an abnormal autoimmune response directed against neuronal or muscular structures of the intestine, even in absence of the previously described autoimmune, infective, or paraneoplastic diseases.The presence of certain antibodies against neuronal or muscle epitopes (anti-Hu, anti-GAD, anti-smooth muscle, anti-gAChR), although not specific, is a useful diagnostic test to suspect these disorders.
Anti-neuronal nuclear antibody type 1 (ANNA-1), also called anti-Hu antibody, is a circulating IgG-class antibody directed against a family of RNA-binding proteins (HuD, HuC, Hel-N1, and Hel-N2) expressed in the nuclei of neurons and small cell lung cancer cells. 64Circulating antibody is detected by immunohistochemistry on brain sections, immunoblots of cortical neurons or recombinant HuD proteins, or enzyme-linked immunosorbent assay of these proteins.7][68][69][70] Based on these data, patients with CIPO should be screened for anti-Hu antibodies, particularly when manometric or histologic evaluations suggest a neuropathic form of CIPO. 63,67,69utamic acid decarboxylase (GAD) is the enzyme that catalyzes the conversion of glutamate to gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the central nervous system.
GAD reduces neuronal excitability in the brain and plays an important role in the regulation of muscle tone.The enzyme is found in all GABA-ergic neurons, but also in pancreatic islet beta cells.Anti-GAD antibodies have been reported in diabetic patients, but also in nondiabetic patients with severe enteric dysmotility, 72 in patients with achalasia, 73 and in a patient with CIPO and dysautonomic symptoms. 74ti-smooth muscle antibodies (ASMA) can be directed against actin, troponin, and tropomyosin.They are increased in autoimmune hepatitis type 1, but have also been reported in a boy who developed CIPO following an episode of gastroenteritis and in whom histologic examination showed T lymphocytic myositis in the circular muscle layer. 75In adult patients, the presence of lymphocytes and plasma cells around smooth muscle cells in the circular muscle layer, in association with replacement of the longitudinal muscle layer with fibrosis, has been reported in two cases 1,76 ; a diffuse CD3 and CD8 lymphocytic infiltrate in both muscle layers has been described in one case 77 ; and a polyclonal lymphoid infiltrate in all muscle layers has been described in four cases. 78The presence of circulating ASMA was not mentioned in these adult patients.
Circulating anti-gAChR correlate with the severity of autonomic dysfunction in patients with autoimmune autonomic neuropathy. 45 one Japanese series, the antibodies were increased in 50% of CIPO patients, but without any significant differences in gastrointestinal symptoms between patients with or without the antibodies. 46

| FULL-THICKNE SS B IOPS IE S
Full-thickness biopsies are usually not recommended in patients with CIPO, as recently reported by an international survey of expert clinicians. 5On the other hand, histologic confirmation of ongoing inflammatory visceral neuropathy or myopathy seems mandatory before starting immunotherapies of uncertain long-term efficacy and with potentially serious side effects. 3In addition, full-thickness biopsies might be indicated in any patient with CIPO and in an overall negative diagnostic workup, since inflammatory forms can be detected in these patients. 79,80The risk-benefit ratio of these indications should be established in each patient.Hopefully, this aspect will be assessed in prospective studies that will take into account the continuous evolution of laparoscopic-assisted 79,81 and endoscopic full-thickness biopsy techniques 80,82 and the yield of standardized methodologies, 83,84 which should be applied in the histological examination.

| G ENE TI C CIP O
CIPO can be one of the clinical manifestations of genetically determined diseases in which a genetic mutation underlies the alteration of muscle, neural, or mesenchymal structures. 37Table 3 reports the genes that have been acknowledged to be involved in CIPO in the online mendelian inheritance in man database (OMIM). 85Genetic mutations are more commonly identified in pediatric than in adult CIPO patients and are often accompanied by functional or anatomical impairment in other organs (bladder, hearth, aorta, brain, dysmorphic features) and this may be a manifestation of genetically determined syndromes. 86However recent studies on family members carrying genetic mutations have shown that mutations previously considered incompatible with life can lead to milder symptoms, reach adulthood, and manifest as sporadic cases. 20,87These studies also suggest that the identification of the phenotype associated to a specific mutation may be difficult according to the extreme variability by which the mutation can be expressed and by the relatively monotonous impact that muscular, neural, or mesenchymal alterations have on gastrointestinal symptoms.In this context, the identification of the anatomical site of gut (mega-esophagus, -stomach, -duodenum, small bowel, -colon, -rectum) or urinary tract (mega-ureter, -bladder) dilatation, such as megaduodenum in Mungan's syndrome 88 or urinary tract dilatation in enteric smooth muscle actin gamma 2 (ACTG2) gene mutations, 89 may help to identify the phenotype.In addition, the concurrent presence of some alterations of esophageal manometry, such as the ineffective or absent contractility in the esophageal body have been found to be associated with ACTG2 mutations affecting smooth muscle function, 20,90 and direct sequencing is recommended in such cases. 20Since the number of phenotypes associated with specific genetic mutations is increasing, genetic testing with parallel massive sequencing, either targeting a pool of selected candidate genes or the whole-exome, is also encouraged when the clinical diagnosis is uncertain.
The most frequent mutations in patients with "primary" CIPO remain those mapping the ACTG2 gene.The first description of this mutation was in 2012 in two adult siblings with visceral myopathy. 91Since then, both de novo and inherited ACTG2 variants in adult patients with CIPO have been described. 20,87Clinical suspicion should arise from the association of CIPO with megacystitis, 92,93 impaired or absent esophageal motility, 20,90 or visceral myopathy on histologic examination. 89,91However, it should be noted that a significant phenotypic variability has been observed between carriers even within the same family.Symptoms of pseudo-obstruction may vary in age of onset, severity, and urinary tract involvement among relatives harboring the same pathogenic mutation.Thus, the possibility of an inherited dominant model, with incomplete penetrance, may endorse the screening of ACTG2 mutations also in asymptomatic parents. 94homozygous missense mutation in RAD21 mapping to chromosome 8 q23-24 have been reported to fully segregate in a Turkish family with megaduodenum, esophageal aperistalsis, Barrett's esophagus, and cardiac abnormalities (Mungan's syndrome).88,95 This is the only mutation identified in RAD21 associated to chronic  pacemaker function of the heart and bowel.Haplotype analysis revealed a founder effect dating back to the 17th century, explaining why all the affected individuals harbor the same genetic variant in SGO1, resulting in the lysine 23 substitution with glutamate at a highly conservative residue.96 Mutations in the X-linked filamin A (FLNA) gene are associated with a wide range of human disorders, globally called filaminopathies, which includes dysmorphic syndromes (otopalatodigital syndromes and frontometaphyseal dysplasia), abnormal neuronal migration (X-linked periventricular heterotopia), vascular and cardiac defects, and intestinal dysmotility with chronic intestinal pseudo-obstruction and X-linked congenital short bowel syndrome.97,98 Differential regulation of the FLNA transcripts explains some of the phenotypic heterogeneity in the filaminopathies, since it has been demonstrated a tissue-specific regulation of FLNA during development, with the long FLNA isoform being predominantly expressed in intestinal smooth muscle, while the short isoform is more abundant in other tissues/organs, including the brain.99 Recent studies on FLNA genetic variants associated with CIPO, demonstrate that a loss-of-function of the longest isoform lead to smooth muscles contractility defects and intestinal elongation deficit.100 Thus, in patients with intestinal complaints without neurological or dysmorphic features, the long isoform of FLNA should be investigated by genetic testing.
Mutations in the MYH11 gene are associated with visceral myopathy, megacystis-microcolon-intestinal hypoperistalsis syndrome 2, and thoracic aortic aneurysm and dissections.It has been hypothesized that the different MYH11-related phenotypes are isoform-specific, with smooth muscle contraction weakness, caused by genetic variants affecting exclusively the SM2 isoform, and loss of elastic fibers and smooth muscle cells in the aorta, caused by pathogenic variants localizing prevalently within the motor domain and the coiled-coil tail domain of SM1. 101cessive megacystis-microcolon-intestinal hypoperistalsis syndrome 1 has been associated also with biallelic loss-of-function variants in MYLK gene, encoding the myosin light chain kinase. 102This kinase phosphorylates myosin light chains to facilitate myosin interaction with actin filaments to produce contractile activity.This gene is expressed both in smooth muscle and non-muscular cells, which explain why heterozygous dominant genetic variants are associated to aortic aneurysm, familial thoracic 7.

| MANOME TRY
12]23 However, this technique is not widely available, 5 it can be limited by the difficult progression of the catheter through the bowel 23 and is often poorly tolerated by patients. 5,10,23An abnormal small-bowel manometry can support the diagnosis of CIPO, which, however, remains based on clinical and radiological findings of a dilated gut. 1,5,11Low amplitude contractions suggest an enteric myopathy but they may also reflect the presence of gut dilatation, while poorly organized contractions of normal amplitude may suggest an enteric neuropathy. 2,79ophageal manometry is an easy-to-perform, available, standardized and usually well tolerated technique that has been found to predict well characterized histological alterations in patients with myopathic 20,90 or neuropathic 67,69 forms of CIPO.Severe esophageal motor abnormalities, such as aperistalsis or achalasia-like alterations, are suggestive of an underlying visceral myopathy or neuropathy, respectively.However, it worth noting that these pathognomonic alterations are found only in a minority of patients. 103orectal manometry can be performed in patients with megacolon, in order to assess the presence of the recto-anal inhibitory reflex after rectal distension. 104The reflex is absent in patients with Hirschsprung's disease and in some CIPO patients with hypo-a-ganglionosis and myenteric ganglionitis. 67,69

| TRE ATMENT
The principles of treatment in patients with CIPO have been reported in detail in previous reviews. 2,3,8Patients with CIPO should be encouraged to eat small and soft frequent meals, according to individual tolerance.Intraluminal fermentation can be reduced through a diet low in lactose and fiber and from courses of antibiotics to control bacterial overgrowth. 8The most commonly used antibiotics are poorly absorbable ones, such as aminoglycosides and rifaximin, as well as metronidazole, amoxicillin clavulanate, doxycycline, and norfloxacin. 4Multivitamin and micronutrient supplementation can be prescribed in order to prevent vitamin and trace elements deficiency. 8Metabolic bone disease should be monitored and prevented. 8,105Drugs that stimulate intestinal motility and tone are useful in only a minority of patients: anticholinesterase inhibitors, such as neostigmine and pyridostigmine, can reduce intestinal dilatation, 106 and prucalopride can improve symptoms, as shown in one of the few controlled studies in patients with CIPO. 107The effect of neostigmine should be evaluated in relation to the increase in abdominal pain often induced by the drug. 106Colonoscopy can be used to decompress an overly distended colon, albeit transiently. 108tomies can provide safe and effective detention of symptomatic segmental bowel distention: percutaneous endoscopic gastrostomy or gastrojejunostomy 109 or percutaneous jejunostomies by interventional radiologist 110 can be performed for decompressive purposes and to facilitate enteral feeding.
Surgery should be avoided except in well-selected cases, as it may increase the risk of adhesions, may reduce the absorbent surface area of the intestine, and may not resolve a functional impairment that often extends throughout the intestine. 3Nutritional support should not be delayed in malnourished patients and in patients who are not able to meet their energy needs with oral nutrition alone and who continue to lose weight.Enteral nutrition is the first step. 8,111A trial of naso-enteric feeding can be performed before placing a permanent enteric feeding tube, in order to assess the optimal composition and rate of the formula.However, enteral nutrition is often not tolerated by patients with severe impairment of small bowel function and parenteral nutrition should be started according to current guidelines. 8,111Intestinal transplantation can be performed in patients with CIPO 112 ; indication in selected cases 8 faces a 5-year mortality rate of 30%. 113psychological and/or psychiatric support may be useful to cope with a chronic and debilitating illness such as CIPO. 1 However, despite the poorer prognosis of patients with CIPO in comparison to patients with enteric dysmotility, a psychological co-morbidity seems less prevalent in the former (9%) than in the latter (56%). 5

| S PECIFI C TRE ATMENT FOR AUTOIMMUNE/INFL AMMATORY CIP O
Immunosuppressive treatments have been attempted in patients with autoimmune/inflammatory CIPO in line with findings in patients with presumed autoimmune gastrointestinal dysmotility, 114 patients with paraneoplastic neurologic syndromes with anti-neuronal antibodies, 115 and patients with autoimmune neuronal disorders. 19Intravenous immunoglobulins or methylprednisolone, have been used as first-line treatment 1,63,66-69,71,76-78 followed by azathioprine, 77 cyclosporine, 66,76 or rituximab 71 in patients with antibodies of suspected pathogenicity or as second-line treatments. 19favorable effect of immunosuppressive treatments on symptoms has often been reported, at least in the short term, but larger studies in well-defined patient subgroups are needed before the long-term efficacy of these approaches can be established.

| G ENE THER APY
Given the wide variety of involved genes and allelic variants responsible for CIPO, it is important for individual patients to have genetic counseling, in order to develop a personalized treatment approach and follow-up based on their specific mutations.Genetic counseling should include psychological support to cope with the diagnosis of a hereditary disease.
Specific gene therapy for CIPO is not yet available, but promising research in this field is ongoing.Gene targeting approaches to visceral myopathies may include gene repairing with CRISPR/ Cas 9-based gene editing or silencing mutations with antisense oligonucleotides. 22Animal models have recently been developed to test these therapeutic strategies.CRISPR/Cas9 editing has been used to generate in vitro and in vivo models in cases where genes are either mutagenized or silenced by inhibiting their expression.Promising results have been obtained from studies on zebrafish models of pediatric intestinal pseudo-obstruction, 116 on organoids from human gastrointestinal tissues, 117 and on induced pluripotent stem cells derived from patients with Hirschsprung's disease. 118enetic testing will certainly be useful in better characterizing the mechanisms underlying motor disorders in patients with CIPO and will help define more homogeneous subgroups of patients.It is hoped that genetic testing will also provide new tools for early diagnosis and targeted genetic therapies of this rare and heterogeneous condition.

| CON CLUS IONS
intestinal pseudo-obstruction until now.Mutations in the shughoshin-1 (SGO1) gene have been found in patients with dilated small and large bowel, sick sinus syndrome with atrial flutter (chronic atrial and bowel dysrhythmia syndrome-CAID), in whom a homozygous recessive mutation in SGO1 impairs TA B L E 3 Genes involved in chronic intestinal pseudo-obstruction and other associated phenotypes acknowledged in the Online Mendelian Inheritance in Man (OMIM).

Figure 1
Figure 1 summarizes the main steps of the diagnostic flowchart in adult patients with CIPO, which aims to: (1) recognize the most frequent diseases that may benefit from specific treatment; (2)identify sporadic inflammatory forms of CIPO that may benefit from immunosuppressive therapies; and (3) consider genetic conditions that may benefit from genetic counseling and, hopefully, in the future of genetic treatment.New studies are needed to evaluate the diagnostic yield of this approach, with particular attention to the indications for full-thickness biopsies in patients with idiopathic CIPO with the suspicion of an underlying autoimmune/inflammatory disease or with an overall negative diagnostic workup.