Lysosomal dysfunction and overload of nucleosides in thymidine phosphorylase deficiency of MNGIE.

Inherited deficiency of thymidine phosphorylase (TP), encoded by TYMP, leads to a rare disease with multiple mitochondrial DNA (mtDNA) abnormalities, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). However, the impact of TP deficiency on lysosomes remains unclear, which are important for mitochondrial quality control and nucleic acid metabolism. Muscle biopsy tissue and skin fibroblasts from MNGIE patients, patients with m.3243 A > G mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) and healthy controls (HC) were collected to perform mitochondrial and lysosomal functional analyses. In addition to mtDNA abnormalities, compared to controls distinctively reduced expression of LAMP1 and increased mitochondrial content were detected in the muscle tissue of MNGIE patients. Skin fibroblasts from MNGIE patients showed decreased expression of LAMP2, lowered lysosomal acidity, reduced enzyme activity and impaired protein degradation ability. TYMP knockout or TP inhibition in cells can also induce the similar lysosomal dysfunction. Using lysosome immunoprecipitation (Lyso- IP), increased mitochondrial proteins, decreased vesicular proteins and V-ATPase enzymes, and accumulation of various nucleosides were detected in lysosomes with TP deficiency. Treatment of cells with high concentrations of dThd and dUrd also triggers lysosomal dysfunction and disruption of mitochondrial homeostasis. Therefore, the results provided evidence that TP deficiency leads to nucleoside accumulation in lysosomes and lysosomal dysfunction, revealing the widespread disruption of organelles underlying MNGIE.

Genome-wide analysis identifies MYH11 compound heterozygous variants leading to visceral myopathy corresponding to late-onset form of megacystis-microcolon-intestinal hypoperistalsis syndrome.

Megacystis-microcolon-hypoperistalsis-syndrome (MMIHS) is a rare and early-onset congenital disease characterized by massive abdominal distension due to a large non-obstructive bladder, a microcolon and decreased or absent intestinal peristalsis. While in most cases inheritance is autosomal dominant and associated with heterozygous variant in ACTG2 gene, an autosomal recessive transmission has also been described including pathogenic bialellic loss-of-function variants in MYH11. We report here a novel family with visceral myopathy related to MYH11 gene, confirmed by whole genome sequencing (WGS). WGS was performed in two siblings with unusual presentation of MMIHS and their two healthy parents. The 38 years-old brother had severe bladder dysfunction and intestinal obstruction, whereas the 30 years-old sister suffered from end-stage kidney disease with neurogenic bladder and recurrent sigmoid volvulus. WGS was completed by retrospective digestive pathological analyses. Compound heterozygous variants of MYH11 gene were identified, associating a deletion of 1.2 Mb encompassing MYH11 inherited from the father and an in-frame variant c.2578_2580del, p.Glu860del inherited from the mother. Pathology analyses of the colon and the rectum revealed structural changes which significance of which is discussed. Cardiac and vascular assessment of the mother was normal. This is the second report of a visceral myopathy corresponding to late-onset form of MMIHS related to compound heterozygosity in MYH11; with complete gene deletion and a hypomorphic allele in trans. The hypomorphic allele harbored by the mother raised the question of the risk of aortic disease in adults. This case shows the interest of WGS in deciphering complex phenotypes, allowing adapted diagnosis and genetic counselling.

Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS): challenges in diagnosis and management.

Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a rare, congenital functional intestinal obstruction, characterised by megacystis (bladder distention in the absence of mechanical obstruction), microcolon and intestinal hypoperistalsis (dysmotility).We are reporting a case of a female child with normal antenatal course who presented with recurrent episodes of abdominal distension since the second day of life and underwent negative exploratory laparotomy on multiple occasions. She also had urinary retention with a grossly distended bladder, requiring drainage by clean intermittent catheterisation. Surgical procedures for bowel decompression, including gastrostomy and ileostomy, were carried out without success. Genetic analysis revealed a mutation in the human smooth muscle (enteric) gamma-actin gene (ACTG2 gene), clinching the diagnosis of MMIHS. The patient was managed with parenteral nutrition and prokinetic medications and tolerated jejunostomy feeds for a brief period before she succumbed to the illness.Female neonates or infants presenting with abdominal distension and dilated urinary tract should be investigated for MMIHS early on. A timely diagnosis will enable the early involvement of a multidisciplinary team to provide the best options available for management.

Chronic intestinal pseudo-obstruction: associations with gut microbiota and genes expression of intestinal serotonergic pathway.

BACKGROUND: Pediatric chronic intestinal pseudo-obstruction (PIPO) is a rare disease characterized by symptoms and radiological signs suggestive of intestinal obstruction, in the absence of lumen-occluding lesions. It results from an extremely severe impairment of propulsive motility. The intestinal endocrine system (IES) jointly with the enteric nervous system (ENS) regulates secreto-motor functions via different hormones and bioactive messengers/neurotransmitters. The neurotransmitter 5-hydroxytryptamine (5-HT) (or serotonin) is linked to intestinal peristalsis and secretory reflexes. Gut microbiota and its interplay with ENS affect 5-HT synthesis, release, and the subsequent serotonin receptor activation. To date, the interplay between 5-HT and gut microbiota in PIPO remains largely unclear. This study aimed to assess correlations between mucosa associated microbiota (MAM), intestinal serotonin-related genes expression in PIPO. To this purpose, biopsies of the colon, ileum and duodenum have been collected from 7 PIPO patients, and 7 age-/sex-matched healthy controls. After DNA extraction, the MAM was assessed by next generation sequencing (NGS) of the V3-V4 region of the bacterial RNA 16 S, on an Illumina Miseq platform. The expression of genes implicated in serotoninergic pathway (TPH1, SLC6A4, 5-HTR3 and 5-HTR4) was established by qPCR, and correlations with MAM and clinical parameters of PIPO have been evaluated. RESULTS: Our results revealed that PIPO patients exhibit a MAM with a different composition and with dysbiosis, i.e. with a lower biodiversity and fewer less connected species with a greater number of non-synergistic relationships, compared to controls. qPCR results revealed modifications in the expression of serotonin-related intestinal genes in PIPO patients, when compared to controls. Correlation analysis do not reveal any kind of connection. CONCLUSIONS: For the first time, we report in PIPO patients a specific MAM associated to underlying pathology and an altered intestinal serotonin pathway. A possible dysfunction of the serotonin pathway, possibly related to or triggered by an altered microbiota, may contribute to dysmotility in PIPO patients. The results of our pilot study provide the basis for new biomarkers and innovative therapies targeting the microbiota or serotonin pathways in PIPO patients.

Generation of CHOPi012-A iPSC line from a patient with visceral myopathy-related chronic intestinal pseudo-obstruction.

Visceral myopathies are debilitating conditions characterized by dysfunction of smooth muscle in visceral organs (bowel, bladder, and uterus). Individuals affected by visceral myopathy experience feeding difficulties, growth failure, life-threatening abdominal distension, and may depend on intravenous nutrition for survival. Unfortunately, our limited understanding of the pathophysiology of visceral myopathies means that current therapies remain supportive, with no mechanism-based treatments. We developed a patient-derived iPSC line with a c.769C > T p.R257C/+ mutation, the most common genetic cause of visceral myopathy. This cell line will facilitate studies of how the ACTG2 R257C heterozygous variant affects smooth muscle development and function.

Distinctive gastrointestinal motor dysfunction in patients with MNGIE.

BACKGROUND: Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare mitochondrial disease caused by mutations in TYMP, encoding thymidine phosphorylase. Clinically it is characterized by severe gastrointestinal dysmotility associated with cachexia and a demyelinating sensorimotor polyneuropathy. Even though digestive manifestations are progressive and invariably lead to death, the features of gastrointestinal motor dysfunction have not been systematically evaluated. The objective of this study was to describe gastrointestinal motor dysfunction in MNGIE using state-of-the art techniques and to evaluate the relationship between motor abnormalities and symptoms. METHODS: Prospective study evaluating gastrointestinal motor function and digestive symptoms in all patients with MNGIE attended at a national referral center in Spain between January 2018 and July 2022. KEY RESULTS: In this period, five patients diagnosed of MNGIE (age range 16-46 years, four men) were evaluated. Esophageal motility by high-resolution manometry was abnormal in four patients (two hypoperistalsis, two aperistalsis). Gastric emptying by scintigraphy was mildly delayed in four and indicative of gastroparesis in one. In all patients, small bowel high-resolution manometry exhibited a common, distinctive dysmotility pattern, characterized by repetitive bursts of spasmodic contractions, without traces of normal fasting and postprandial motility patterns. Interestingly, objective motor dysfunctions were detected in the absence of severe digestive symptoms. CONCLUSIONS AND INFERENCES: MNGIE patients exhibit a characteristic motor dysfunction, particularly of the small bowel, even in patients with mild digestive symptoms and in the absence of morphological signs of intestinal failure. Since symptoms are not predictive of objective findings, early investigation is indicated.

Pediatric Intestinal Pseudo-Obstruction: An International Survey on Diagnostic and Management Strategies in the European Reference Network for Rare Inherited and Congenital Anomalies Intestinal Failure Teams.

OBJECTIVES: Pediatric intestinal pseudo-obstruction (PIPO) management is based on nutritional, medical, and surgical care while available evidence is scarce. The aim of this study was to outline the current diagnostic and management strategies in intestinal failure (IF) teams of the European Reference Network for rare Inherited and Congenital Anomalies (ERNICA) and to compare these practices to the latest PIPO international guidelines. METHODS: An online survey on institutional diagnostic and management strategies of PIPO was conducted among the ERNICA IF teams. RESULTS: In total, 11 of 21 ERNICA IF centers from 8 countries participated. On average, 64% of teams had ≥6 and 36% had 1-5 PIPO patients under active follow-up. In total, 80 of 102 PIPO patients were parenteral nutrition (PN) dependent while each IF team had median 4 (range 0-19) PN dependent PIPO patients under follow-up. On average, each center received 1-2 new PIPO patients per year. Diagnostics mostly followed current guidelines while medical and surgical management strategies were diverse. CONCLUSIONS: Numbers of PIPO patients are low and management strategies are diverse among ERNICA IF teams. To improve PIPO patient care, regional reference centers with specialized multidisciplinary IF teams and continuous collaboration across centers are needed.

Patient's dermal fibroblasts as disease markers for visceral myopathy.

Visceral myopathy (VSCM) is a rare genetic disease, orphan of pharmacological therapy. VSCM diagnosis is not always straightforward due to symptomatology similarities with mitochondrial or neuronal forms of intestinal pseudo-obstruction. The most prevalent form of VSCM is associates with variants in the gene ACTG2, encoding the protein gamma-2 actin. Overall, VSCM is a mechano-biological disorder, in which different genetic variants lead to similar alterations to the contractile phenotype of enteric smooth muscles, resulting in the emergence of life-threatening symptoms. In this work we analyzed the morpho-mechanical phenotype of human dermal fibroblasts from patients affected with VSCM, demonstrating that they retain a clear signature of the disease when compared with different controls. We evaluated several biophysical traits of fibroblasts, and we show that a measure of cellular traction forces can be used as a non-specific biomarker of the disease. We propose that a simple assay based on traction forces could be designed to provide a valuable support for clinical decision or pre-clinical research.

Histopathological, Ultrastructural, and Immunohistochemical Findings in MYH11-Variant Visceral Myopathy.

BACKGROUND: Pathogenic mutations in the smooth muscle myosin heavy chain gene, MYH11, cause megacystis megacolon intestinal hypoperistalsis syndrome and other forms of chronic intestinal pseudo-obstruction. Evaluation of intestinal tissues from affected patients is often performed before mutational analysis, but the pathological findings of MYH11-variant visceral myopathy have not been well defined. METHODS: Light microscopic, immunohistochemical, and ultrastructural findings from multiple intestinal samples from 2 patients with MYH11-variant visceral myopathy were reviewed, including MYH11-specific immunohistochemistry. The findings were compared with intestinal samples from patients with gamma-smooth muscle actin (ACTG2)-variant visceral myopathy and non-pseudo-obstruction controls. RESULTS: Apart from non-specific changes (e.g., muscle hypertrophy and distension-related muscularis propria necrosis), no alterations were identified by routine histopathological evaluation or electron microscopy. Immunohistochemistry with antibodies against a battery of smooth muscle proteins, including MYH11, revealed indistinguishable patterns of immunoreactivity in the muscularis propria of both patients and controls. CONCLUSIONS: Myopathic morphological or immunohistochemical changes may not be present in intestinal specimens from patients with MYH11-variant visceral myopathy. Molecular genetic studies should be considered for patients with chronic intestinal pseudo-obstruction and normal or non-specific pathology findings.

Heterozygous Actg2R257C mice mimic the phenotype of megacystis microcolon intestinal hypoperistalsis syndrome.

BACKGROUND: Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a rare and serious congenital disorder with poor outcomes, where a heterozygous missense mutation is present in the ACTG2 gene. Here, we aimed to investigate the pathogenesis of ACTG2 in MMIHS. METHODS: A cohort with 20 patients with MMIHS was screened. Actg2R257C heterozygous mutant mice were generated using the CRISPR/Cas9 system. Gastrointestinal (GI) motility, voluntary urination, collagen gel contraction, and G-actin/F-actin analysis were performed. KEY RESULTS: The R257C variant of ACTG2 most frequently occurred in patients with MMIHS and demonstrated the typical symptoms of MMIHS. Actg2R257C heterozygous mutant mice had dilated intestines and bladders. The functional assay showed a prolonged total time of GI transit and decreased urine spot area. Collagen gel contraction assay and G-actin/F-actin analysis indicated that mutant mice showed reduced area of contraction of smooth muscle cells (SMCs) and impaired actin polymerization. CONCLUSIONS & INFERENCES: A mouse model demonstrating MMIHS-like symptoms was generated. The Actg2R257C heterozygous variant impairs SMCs contraction by interfering with actin polymerization, leading to GI motility disorders.

The long Filamin-A isoform is required for intestinal development and motility: implications for chronic intestinal pseudo-obstruction.

Filamin A (FLNA) is a cytoplasmic actin binding protein, recently shown to be expressed as a long and short isoform. Mutations in FLNA are associated with a wide spectrum of disorders, including an X-linked form of chronic intestinal pseudo-obstruction (CIPO). However, the role of FLNA in intestinal development and function is largely unknown. In this study, we show that FLNA is expressed in the muscle layer of the small intestine from early human fetal stages. Expression of FLNA variants associated with CIPO, blocked expression of the long flna isoform and led to an overall reduction of RNA and protein levels. As a consequence, contractility of human intestinal smooth muscle cells was affected. Lastly, our transgenic zebrafish line showed that the flna long isoform is required for intestinal elongation and peristalsis. Histological analysis revealed structural and architectural changes in the intestinal smooth muscle of homozygous fish, likely triggered by the abnormal expression of intestinal smooth muscle markers. No defect in the localization or numbers of enteric neurons was observed. Taken together, our study demonstrates that the long FLNA isoform contributes to intestinal development and function. Since loss of the long FLNA isoform does not seem to affect the enteric nervous system, it likely results in a myopathic form of CIPO, bringing new insights to disease pathogenesis.

Enteric Neuromyopathies: Highlights on Genetic Mechanisms Underlying Chronic Intestinal Pseudo-Obstruction.

Severe gut motility disorders are characterized by the ineffective propulsion of intestinal contents. As a result, the patients develop disabling/distressful symptoms, such as nausea and vomiting along with altered bowel habits up to radiologically demonstrable intestinal sub-obstructive episodes. Chronic intestinal pseudo-obstruction (CIPO) is a typical clinical phenotype of severe gut dysmotility. This syndrome occurs due to changes altering the morpho-functional integrity of the intrinsic (enteric) innervation and extrinsic nerve supply (hence neuropathy), the interstitial cells of Cajal (ICC) (mesenchymopathy), and smooth muscle cells (myopathy). In the last years, several genes have been identified in different subsets of CIPO patients. The focus of this review is to cover the most recent update on enteric dysmotility related to CIPO, highlighting (a) forms with predominant underlying neuropathy, (b) forms with predominant myopathy, and (c) mitochondrial disorders with a clear gut dysfunction as part of their clinical phenotype. We will provide a thorough description of the genes that have been proven through recent evidence to cause neuro-(ICC)-myopathies leading to abnormal gut contractility patterns in CIPO. The discovery of susceptibility genes for this severe condition may pave the way for developing target therapies for enteric neuro-(ICC)-myopathies underlying CIPO and other forms of gut dysmotility.

Clinical and Pathological Features of Severe Gut Dysmotility.

Severe gut motility disorders are characterized by ineffective propulsion of intestinal contents. As a result, patients often develop extremely uncomfortable symptoms, ranging from nausea and vomiting along with alterations of bowel habits, up to radiologically confirmed subobstructive episodes. Chronic intestinal pseudo-obstruction (CIPO) is a typical clinical phenotype of severe gut dysmotility due to morphological and functional alterations of the intrinsic (enteric) innervation and extrinsic nerve supply (hence neuropathy), interstitial cells of Cajal (ICCs) (mesenchymopathy), and smooth muscle cells (myopathy). In this chapter, we highlight some molecular mechanisms of CIPO and review the clinical phenotypes and the genetics of the different types of CIPO. Specifically, we will detail the role of some of the most representative genetic mutations involving RAD21, LIG3, and ACTG2 to provide a better understanding of CIPO and related underlying neuropathic or myopathic histopathological abnormalities. This knowledge may unveil targeted strategies to better manage patients with such severe disease.

Variant in ACTG2 Causing Megacystis Microcolon Hypoperistalsis Syndrome and Severe Familial Postpartum Bleeding.

INTRODUCTION: Megacystis microcolon hypoperistalsis syndrome (MMIHS) is a rare condition with high morbidity and mortality. It is characterized by megacystis, microcolon, and intestinal hypoperistalsis leading to various grades of bladder and bowel obstruction. CASE PRESENTATION: This report describes a pregnant woman with a history of bowel obstruction, urine retention, and heavy postpartum bleeding where ultrasound findings of fetal megacystis during pregnancy led to genetic testing in the family. The fetus, the pregnant woman, and four female family members were heterozygous for a pathogenic variant detected in the ACTG2 gene. The fetus was treated successfully for hydronephrosis using vesicoamniotic shunting. DISCUSSION: Early diagnosis of a fetus with MMIHS is important to secure multidisciplinary prenatal and neonatal treatment. Furthermore, gene testing must be considered when a woman presents a history of pseudo-obstruction and urine retention to prevent complications during pregnancy and labor. Finally, recurrent familial postpartum bleeding should lead to referral to genetic evaluation.

Intestinal Pathology in Patients With Pathogenic ACTG2-Variant Visceral Myopathy: 16 Patients From 12 Families and Review of the Literature.

BACKGROUND: Dominant gamma-smooth muscle actin gene (ACTG2) variants cause clinically diverse forms of visceral myopathy. Many patients undergo intestinal resection or biopsy before identification of their genetic defect. The pathology of ACTG2-variant visceral myopathy has not been evaluated systematically. METHODS: Glass slides, ultrastructural images, molecular genetic reports, and clinical records from 16 patients with pathogenic (15) or likely pathogenic (1) ACTG2 variants were reviewed and compared with surgical specimens from controls (no evidence of a primary myopathy or pseudo-obstruction due to Hirschsprung disease) and published descriptions. RESULTS: The variable clinical manifestations in our cohort matched those in the literature. Only non-specific light and electron microscopic findings observed in non-myopathic controls were encountered in 13 of 16 patients. The remaining 3 patients harbored hyalinized cytoplasmic inclusions in smooth muscle cells and 1 of them had polyglucosan bodies in the muscularis propria. CONCLUSIONS: Apart from hyalinized inclusions, which were only observed in 3/16 patients, intestinal pathology in the majority of patients with ACTG2 variants is not indicative of an underlying visceral myopathy. Molecular testing should be considered even when no diagnostic intestinal pathology is identified.

[Familial intestinal degenerative neuropathy with chronic pseudo-obstruction linked to duplication in chromosome 9]. / Helsingborgssjukan kopplad till duplikation i kromosom 9.

The so-called Helsingborg Disease is an intestinal degenerative neuropathy discovered in a kindred in South Sweden. Affected subjects develop severe intestinal symptoms, the most common being chronic diarrhea, but abdominal pain, constipation and severe vomiting are common. Chronic intestinal pseudo-obstruction is the end-stage with high mortality from intestinal failure. Two families with affected members were shown by histopathology and genealogy to be one large kindred with the same underlying disease. Genetic analysis shows that this disease, having an autosomal dominant transmission, is strongly linked to a region in the short arm of chromosome 9 and a 1.2 Mb duplication in this region. The duplication includes 22 protein-coding genes, most of them are interferon genes. Family members not carrying the duplication have no increased prevalence of gut diseases. Genetic analysis including gene-dose array should be important for etiologic diagnosis and for genetic guidance in this kindred.

Loss-of-function variants within LMOD1 actin-binding site 2 cause pediatric intestinal pseudo-obstruction by impairing protein stability and actin nucleation.

The leiomodin1 (LMOD1) gene, encoding a potent actin nucleator, was recently reported as a potential pathogenic gene of megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS, OMIM 619362). However, only a single patient has been reported to have LMOD1 mutations, and the underlying pathogenic mechanism remains unknown. Here, we described a male infant with LMOD1 mutations presenting typical symptoms of pediatric intestinal pseudo-obstruction (PIPO) but without megacystis and microcolon. Two compound heterozygous missense variants (c.1106C>T, p.T369M; c.1262G>A, p.R421H) were identified, both affecting highly conserved amino acid residues within the second actin-binding site (ABS2) domain of LMOD1. Expression analysis showed that both variants resulted in significantly reduced protein amounts, especially for p.T369M, which was almost undetectable. The reduction was only partially rescued by the proteasome inhibitor MG-132, indicating that there might be proteasome-independent pathways involved in the degradation of the mutant proteins. Molecular modeling showed that variant p.T369M impaired the local protein conformation of the ABS2 domain, while variant p.R421H directly impaired the intermolecular interaction between ABS2 and actin. Accordingly, both variants significantly damaged LMOD1-mediated actin nucleation. These findings provide further human genetic evidence supporting LMOD1 as a pathogenic gene underlying visceral myopathy including PIPO and MMIHS, strengthen the critical role of ABS2 domain in LMOD1-mediated actin nucleation, and moreover, reveal an unrecognized role of ABS2 in protein stability.

LPAR1 regulates enteric nervous system function through glial signaling and contributes to chronic intestinal pseudo-obstruction.

Gastrointestinal motility disorders involve alterations to the structure and/or function of the enteric nervous system (ENS) but the causal mechanisms remain unresolved in most cases. Homeostasis and disease in the ENS are processes that are regulated by enteric glia. Signaling mediated through type I lysophosphatidic acid receptors (LPAR1) has recently emerged as an important mechanism that contributes to disease, in part, through effects on peripheral glial survival and function. Enteric glia express LPAR1 but its role in ENS function and motility disorders is unknown. We used a combination of genetic, immunohistochemical, calcium imaging, and in vivo pharmacological approaches to investigate the role of LPAR1 in enteric glia. LPAR1 was enriched in enteric glia in mice and humans and LPA stimulated intracellular calcium responses in enteric glia, subsequently recruiting activity in a subpopulation of myenteric neurons. Blocking LPAR1 in vivo with AM966 attenuated gastrointestinal motility in mice and produced marked enteric neuro- and gliopathy. Samples from humans with chronic intestinal pseudo-obstruction (CIPO), a severe motility disorder, showed reduced glial LPAR1 expression in the colon and ileum. These data suggest that enteric glial LPAR1 signaling regulates gastrointestinal motility through enteric glia and could contribute to severe motility disorders in humans such as CIPO.