As previously outlined, CIPO is a very severe form of gut dysmotility characterized by recurrent sub-obstructive episodes in the absence of any evidence of mechanical causes occluding the intestinal lumen ^[1][2][1,14]. The classification of CIPO is based on three major subtypes: (i) ‘‘secondary’’ forms, i.e., those cases related to a wide array of recognized pathological conditions; (ii) ‘‘idiopathic’’ forms, i.e., cases with unknown etiology; and, finally, (iii) “primary” forms, which can be applied to patients with a possible genetic origin. So far, the management of CIPO patients remains largely unsatisfactory, thus leading to frustration among the patients, their families, and physicians.

In-depth in vitro and in vivo studies of gene variants are required to understand their impact in generating the severe enteric dysmotility experienced by CIPO patients. In this context, the discovery of the mutated genes represents the first step for developing novel targeted therapeutic strategies aimed at overcoming downstream molecular impairments.

The neuropathological findings reported in neurodegenerative CIPO cases include various qualitative (neuronal swelling, intranuclear inclusions, axonal degeneration, and other lesions) and quantitative (oligoneuronal hypoganglionosis) abnormalities of the ENS ^[1][3][1,34]. Sporadic cases of enteric neuropathies are associated with two major patterns of abnormalities: (a) a marked reduction in intramural (especially myenteric) neuronal cells mainly associated with swollen neural cell bodies and processes, the fragmentation and loss of axons, and the proliferation of glial cells, and (b) a loss of normal staining in subsets of enteric neurons in the absence of dendritic swelling or glial proliferation [1]. Full-thickness specimens from patients with CIPO indicated that symptoms/clinical manifestations and severity increase as the number of enteric neurons decreases. Compared to control tissues, a 50% loss of neuronal cells in the myenteric and submucosal ganglia may be a “critical threshold” for recurrent sub-obstructive episodes, small bowel dilatation, and other severe symptoms ^[3][34]. Different findings have been identified in biopsies of the GI tract from patients with Parkinson’s disease (PD) or diabetes. Neuropathological and ICC changes have been detected in the gastric neuromuscular layer of patients with diabetic gastroparesis/gastroenteropathy ^[4][35]. Lewy pathology (i.e., intraneuronal deposits of phosphorylated α-synuclein) can be visualized in the ENS of patients with PD before a clinical diagnosis is established. This feature supports the notion that the GI tract exerts a key role in the pathogenesis of PD ^[5][36]. These abnormalities may likely involve the ENS and ICC more diffusely throughout the gut, but consistent data regarding CIPO related to diabetes mellitus and PD are still lacking.

In recent years, reseaourchers' team and other groups have provided evidence of a genetic basis for the enteric neuronal degeneration and loss observed in specific forms of CIPO. The discovery of novel genes mutated in different patients represents the first step in identifying the cause of the downstream molecular impairment in CIPO. Homozygous mutations in SGO1 and RAD21, which code for cohesin complex components, were identified in patients with CIPO ^[6][37]. Chetaille et al. described a new syndrome caused by a SGO1 mutation affecting shugoshin-1’s structure and function. The authors defined this condition as chronic atrial and intestinal dysrhythmia (CAID) syndrome, i.e., a novel generalized dysrhythmia, indicative of the role of SGO1 in mediating the integrity of human cardiac and gut rhythms, the latter being generated by ICC. Since shughoshin-1 (SGO1) is part of the cohesin complex, its dysfunction could result in consequences for long-range transcriptional regulation, possibly interfering with the expression of genes associated with CIPO ^[7][32]. Indeed, a recent study showed that an impaired inward rectifier potassium current, alterations of canonical TGF-beta signaling, and epigenetic dysregulation contributed to the development of the intestinal and cardiac manifestations observed in CAID syndrome ^[8][38].

RWesearchers identified the homozygous causative variant in a large consanguineous family segregating an autosomal recessive form of CIPO in the Double-Strand-Break Repair Protein Rad21 Homolog (RAD21) gene. In the affected family members, other clinical features included a megaduodenum, a long-segment Barrett esophagus (up to 18 cm from the “Z-line”), and cardiac (interventricular septum) abnormalities of variable severity (OMIM 611376, also referred to as Mungan syndrome). RWesearchers performed a whole-exome-sequencing analysis on the genomic DNA from two affected individuals and found a novel homozygous change, namely, c.1864 G>A in RAD21, responsible for damaging the missense variant p.Ala622Thr ^[9][30]. Any dysfunction of RAD21’s molecular structure and function can result in significant changes in many tissues, including the gut neuro-muscular layer. In fact, RAD21 is part of the cohesin complex involved in the pairing and unpairing of sister chromatids during cell replication and division, as well as in regulating gene expression directly and independently of cell division ^[10][39]. The RAD21 subunit of the cohesin complex plays important structural and functional roles, as it serves as a physical link between the Structural Maintenance of Chromosome 1 (SMC1) and 3 (SMC3) heterodimers and the Stromal Antigen (STAG) subunit. RAD21 integrity regulates the association or disassociation of functional cohesin with chromatin and has a key role in double-strand break DNA repair ^[11][12][40,41]. In in vivo experiments using a zebrafish model, reswearchers recapitulated the CIPO phenotype observed in patients with the homozygous RAD21 variant showing a severe impairment of motility and a marked reduction in the pan-neuronal marker HuC/D-immunoreactive enteric neurons, a finding reminiscent of an oligo-neuronal hypoganglionosis detected in CIPO patients ^[9][30]. Further studies using novel mouse models will help elucidate the molecular pathways altered by RAD21 genetic defects.

A recent study by Le et al. (2021 ^[13][42]) showed that mutations in the genes coding for Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2) and Erb-B3 Receptor Tyrosine Kinase 3 (ERBB3) led to a broad spectrum of developmental abnormalities, including intestinal dysmotility. A thorough gut histology assessment revealed aganglionosis, hypoganglionosis, and intestinal smooth muscle abnormalities in the affected patients. The cell type-specific ErbB3 and ErbB2 functions were determined through single-cell RNA sequencing data in a conditional ErbB3-deficient mouse model, which revealed a central role for ErbB3 in enteric progenitors. Further mechanistic investigations will improve the understanding of the role of ErbB3/ErbB2 pathways in ENS development, maintenance, and disease states ^[13][14][21,42].

Visceral myopathies are characterized by smooth muscle cell abnormalities and are most commonly caused by mutations in the contractile apparatus cytoskeletal proteins (for an extensive review, see ^[15][43]), such as ACTG2 ^[16][17][16,20], ACTA2 ^[18][19], MYH11 ^[19][20][26,44], MYLK ^[21][28], LMOD1 ^[22][25], MYL9 ^[23][45], and FLNA ^[24][25][26][22,23,46]. Visceral myopathy (MIM# 155310) due to ACTG2 pathogenic variants causes gut dysmotility due to smooth muscle dysfunction with phenotypes ranging from cases predominantly affecting the GI tract with typical CIPO features to the megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS), which is characterized by severely disabling defects, such as dysfunctional bowels, reduced intrauterine colon growth (microcolon), and bladder-emptying defects requiring catheterization ^[15][43]. MMIHS can be considered a severe form of CIPO in early infancy with affected patients having a poor prognosis and short life expectancy.

In patients whose bowels are mainly affected and a microcolon is absent, the condition can be labeled as myopathic CIPO. Causative heterozygous variants in ACTG2 result in dominant disorders running in families or arising de novo in the affected subjects ^[17][20]. In a large study, the rate of molecular diagnosis in visceral myopathy cases was 64%, of which 97% was due to ACTG2 variants. Missense changes in five conserved arginine residues of ACTG2 contributed to 49% of cases [16]. The ACTG2-negative patients had a more favorable clinical outcome and more restricted disease. In the ACTG2-positive group, the poor outcome (i.e., total parenteral nutrition dependence, the need for transplantation, and death) was always associated with one of the arginine missense alleles. The analysis of the effect of the specific residues suggested the degree of severity of the missense changes, with substitutions at p.Arg178 exerting a more damaging effect than substitutions at p.Arg257 and p.Arg40, along with other less frequent variant alleles at p.Arg63 and p.Arg211. Four novel missense variants were also reported, including one transmitted according to a recessive mode of inheritance ^[27][47], indicating that the full genetic architecture of visceral myopathy has still to be fully characterized. Interestingly, in a recent review, Fournier and Fabre evaluated the mutation frequency observed in the genes involved in visceral myopathy in 117 published cases (112 patients and 5 pregnancies ended before birth) ^[28][48]. In concordance with previous studies, the most frequently reported mutations were in ACTG2 (75/112, 67% of patients), MYH11 (14%), and FLNA (13%).

It is worth noting that some patients with pathogenic ACTG2 mutations develop the disease later in life and survive to adulthood without needing parenteral nutrition; conversely, others with the same ACTG2 mutation may exhibit a severe form of the disease in childhood. Such variability in symptom severity in individuals with the same ACTG2 gene defect strongly suggests that other factors, genetic or non-genetic, beyond the causative variants can impact the clinical phenotype. Understanding the reasons why clinical phenotypes vary despite identical ACTG2 mutations may lead to new therapeutic strategies for these myopathic forms of CIPO.

The prevailing hypothesis is that ACTG2 mutations cause smooth muscle abnormalities by disrupting the contractile cytoskeletal protein apparatus. In vitro studies in transfected cells showed an impairment of ACTG2 polymerization and a reduction in smooth muscle cell contractility in the presence of the mutant form ^[21][28]. A recent study by Hashmi et al. demonstrated that in primary human intestinal smooth muscle cells (HISMCs) the ACTG2^R257C mutation profoundly alters the ACTG2 filament bundle structure, generating less robust fibers without altering the global actin cytoskeleton. Notably, ACTG2^R257C-expressing HISMCs spread and migrated faster than the wild-type ones, suggesting that the mutation induces a less differentiated and less functional status in enteric smooth muscle cells ^[29][49].

Additional genes have been found to play a role in visceral myopathy pathogenesis. ACTA2, coding for a smooth muscle actin gene, is mutated in the multisystemic smooth muscle dysfunction syndrome (MSMDS; MIM #613834). The clinical features include bladder hypotonicity, abnormal intestinal peristalsis, and the prominent involvement of vascular and ciliary smooth muscles, leading to vascular aneurysms and mydriasis ^[18][19]. Different autosomal recessive forms of MMIHS are caused by biallelic loss-of-function variants in genes coding for proteins involved in actin–myosin interactions, such as MYH11 (myosin heavy chain; ^[19][26], MYLK (myosin-light chain kinase; ^[21][28], LMOD1 (leiomodin 1, an actin-binding protein expressed primarily in vascular and visceral smooth muscle ^[21][28]; and MYL9 (regulatory myosin-light chain) ^[23][45]). Several studies have identified alterations in smooth muscle structural proteins and pathways related to smooth muscle function, providing substantial molecular insights into the disease’s pathogenesis. As an example, the loss of LMOD1 in vitro and in vivo results in a reduction in filamentous actin, generating elongated cytoskeletal dense bodies and impairing intestinal smooth muscle contractility ^[21][28].

Mutations in the X-linked gene FLNA were previously only associated with forms of a neuropathic origin, but detailed immunohistochemical analysis has demonstrated diffuse, abnormal layering of the intestinal smooth muscle with no enteric neuron involvement ^[26][46]. FLNA has two isoforms—the longer one is predominant in the intestinal smooth muscle, and mutations in this isoform cause CIPO with periventricular nodular heterotopia in the brain ^[30][50].

Finally, myopathic forms of severe gut dysmotility have been identified in patients affected by myotonic dystrophy type 1, Duchenne muscular dystrophy, and oculo-gastrointestinal muscular dystrophy ^[31][51]. Scleroderma, systemic lupus erythematosus, dermatomyositis, polymyositis, amyloidosis, and ceroidosis can also cause myopathic CIPO ^[32][7].