Diagnostic yield and novel candidate genes by next generation sequencing in 166 children with intrahepatic cholestasis.

BACKGROUND AND AIMS: Cholestatic liver disease is a leading referral to pediatric liver transplant centers. Inherited disorders are the second most frequent cause of cholestasis in the first month of life. METHODS: We retrospectively characterized the genotype and phenotype of 166 participants with intrahepatic cholestasis, and re-analyzed phenotype and whole-exome sequencing (WES) data from patients with previously undetermined genetic etiology for newly published genes and novel candidates. Functional validations of selected variants were conducted in cultured cells. RESULTS: Overall, we identified disease-causing variants in 31% (52/166) of our study participants. Of the 52 individuals, 18 (35%) had metabolic liver diseases, 9 (17%) had syndromic cholestasis, 9 (17%) had progressive familial intrahepatic cholestasis, 3 (6%) had bile acid synthesis defects, 3(6%) had infantile liver failure and 10 (19%) had a phenocopy of intrahepatic cholestasis. By reverse phenotyping, we identified a de novo variant c.1883G > A in FAM111B of a case with high glutamyl transpeptidase (GGT) cholestasis. By re-analyzing WES data, two patients were newly solved, who had compound heterozygous variants in recently published genes KIF12 and USP53, respectively. Our additional search for novel candidates in unsolved WES families revealed four potential novel candidate genes (NCOA6, CCDC88B, USP24 and ATP11C), among which the patients with variants in NCOA6 and ATP11C recapitulate the cholestasis phenotype in mice models. CONCLUSIONS: In a single-center pediatric cohort, we identified monogenic variants in 22 known human intrahepatic cholestasis or phenocopy genes, explaining up to 31% of the intrahepatic cholestasis patients. Our findings suggest that re-evaluating existing WES data from well-phenotyped patients on a regular basis can increase the diagnostic yield for cholestatic liver disease in children.

Roxadustat ameliorates experimental colitis in mice by regulating macrophage polarization through increasing HIF level.

BACKGROUND: Gastro-intestinal (GI) tract inflammation is as a result of inflammatory hypoxia which is also induced by long-standing group of disorders like inflammatory-bowel disease (IBD). Regulation of GI immune homeostasis by macrophage involves hypoxia-inducible factor (HIF). As inhibitor of HIF prolyl hydroxylase, roxadustat (ROX) increases the levels of HIF. METHODS: We induced experimental colitis (EC) model in mice via dextran-sulfate sodium (DSS) to evaluate ROX role in above-mentioned disease. RESULTS: ROX ameliorated EC in mice by blocking colonic length shorten and loss of body weight, thereby reducing scores of disease-activity index (DAI) and histopathology. ROX significantly reduced inflammatory cytokines levels, suppressed M1 and increased M2 macrophage polarization in colonic tissues. Besides, ROX blocked declining hematocrit (HCT) level in blood and increased HIF-1-α and HIF-2-α level in colonic tissues. The inhibitor of HIF-1- α, KC7F2 decreased body weight and colonic length in ROX-treated DSS mice. Meanwhile, DAI scores and histopathology in KC7F2 treated DSS mice were markedly higher than that of treatment with ROX alone. KC7F2 treatments also significantly increased inflammatory cytokines levels, respectively promoted and reduced polarization of M1 and M2 macrophages in colonic tissue from ROX treated mice. Further, KC7F2 treatments inhibited ROX induced HCT level increasing in blood and decreased HIF-1-α and HIF-2-α level in colonic tissue. CONCLUSION: Collectively, we discovered that ROX ameliorated EC in mice by regulating macrophage polarization through promotion of HIF expression. GENERAL SIGNIFICANCE: Taken together, we developed a new application of ROX, which provides new ideas and a scientific basis for IBD treatment.

Acute liver failure and aplastic crisis due to anorexia nervosa in an adolescent girl: a case report.

Anorexia nervosa (AN) has a high mortality rate due to the widespread organ dysfunction caused by the underlying severe malnutrition. Malnutrition-induced hepatitis is common among individuals with AN especially as body mass index decreases, while acute liver failure and aplastic crisis related to coagulation disease and encephalopathy rarely occur in AN patients. The supervised increase of caloric intake can quickly improve the elevated aminotransferases caused by starvation and aplastic crisis. This current case report describes a 12-year-old adolescent girl who was admitted with a 3-month history of weight loss. Within 3 months, she had lost 10 kg of weight. The girl was diagnosed with AN, acute liver failure, severe malnutrition with emaciation, electrolyte disorder, bradycardia and aplastic crisis. She was gradually supplemented with vitamins and enteral nutrition to avoid refeeding syndrome. After treatment, her liver function and haematopoietic function returned to normal. In conclusion, acute liver failure and aplastic crisis are rare but potentially life-threatening complications of AN, which could be improved by supervised feeding and timely rehydration. AN should be considered as the potential aetiology of acute liver failure and aplastic crisis.

Characteristics of the Cajal interstitial cells and intestinal microbiota in children with refractory constipation.

BACKGROUND: Children with refractory constipation experience intense and persistent symptoms that greatly diminish their quality of life. However, the underlying pathophysiological mechanism responsible for this condition remains uncertain. Our objective was to evaluate characteristics of colonic motor patterns and interstitial cells of Cajal (ICCs) to refractory constipation children, as well as intestinal microbiota compositions. METHODS: Colonic manometry (CM) was conducted on a cohort of 30 patients with refractory constipation to assess colonic motility, and 7 of them underwent full-thickness colon biopsy specimens. Another 5 colonic specimens from nonconstipation patients were collected to identify the ICCs by immunohistochemistry. Fecal samples from 14 children diagnosed with refractory constipation and subjecting 28 age-matched healthy children to analysis using high-throughput sequencing of 16S rRNA. RESULTS: According to CM results, dividing 30 children with refractory constipation into 2 groups: normal group (n = 10) and dysmotility group (n = 20). Dysmotility subjects showed lower colonic motility. Antegrade propagating pressure waves, retrograde propagating pressure waves, and periodic colonic motor activity were common in normal subjects and rare in dysmotility subjects (32.7 ± 8.9 vs 20.7 ± 13.0/17 h, P < 0.05, 11.5 ± 2.3 vs 9.6 ± 2.3/17 h, P < 0.05, and 5.2 ± 8.9 vs 3.5 ± 6.8 cpm, P < 0.005, respectively), whereas periodic rectal motor activity was more common in dysmotility subjects (3.4 ± 4.8 vs 3.0 ± 3.1 cpm, P < 0.05). Dysmotility subjects exhibited a significantly greater number of preprandial simultaneous pressure waves compared to the normal subjects (32.3 ± 25.0 vs 23.6 ± 13.2/1 h, P < 0.005). Dysmotility subjects displayed a notable decrease in postprandial count of antegrade propagating pressure waves and high amplitude propagating pressure waves when compared to normal subjects (3.9 ± 2.9 vs 6.9 ± 3.5/1 h and 2.3 ± 1.5 vs 5.4 ± 2.9/1 h, respectively, P < 0.05). The number, distribution, and morphology of ICCs were markedly altered in refractory constipation compared children to the controls (P < 0.05). Children diagnosed with refractory constipation displayed a distinct dissimilarity in composition of their intestinal microbiota comparing with control group (P < 0.005). In genus level, Bacteroidetes represented 34.34% and 43.78% in the refractory constipation and control groups, respectively. Faecalibacterium accounted for 3.35% and 12.56%, respectively (P < 0.005). Furthermore, the relative abundances of Faecalibacterium (P < 0.005), Lachnospira (P < 0.05), and Haemophilus (P < 0.05) significantly decreased, whereas those of Parabacteroides (P < 0.05), Alistipes (P < 0.005), Prevotella_2 (P < 0.005), [Ruminococcus]_torques_group (P < 0.005), Barnesiella (P < 0.05), Ruminococcaceae_UCG-002 (P < 0.005), and Christensensenellaceae_R-7_group (P < 0.05) were markedly increased in children with refractory constipation. CONCLUSIONS: Dysmotility subjects showed lower colonic motility and an impaired postprandial colonic response. The decreased number and abnormal morphology of colonic ICCs may contribute to the pathogenesis of refractory constipation. Children with refractory constipation exhibited significant variations in microbiota composition across various taxonomic levels compared to the healthy control group. Our findings contribute valuable insights into pathophysiological mechanism underlying refractory constipation and provide evidence to support the exploration of novel therapeutic strategies for affected children.

Transient pseudohypoaldosteronism in infancy mainly manifested as poor appetite and vomiting: Two case reports and review of the literature.

Introduction: Transient Pseudohypoaldosteronism (TPHA) is a very rare condition usually secondary to urinary tract malformations (UTM) and/or urinary tract infection (UTI). It is characterized by hyperkalemia, hyponatremia, metabolic acidosis, and elevated plasma aldosterone levels. Given that the predominant manifestations of TPHA patients are digestive tract symptoms, such as poor appetite, vomiting, and weight gain, it is easily misdiagnosed as digestive tract diseases. Case reports: Two children with poor appetite and vomiting were admitted to the Department of Gastroenterology, Children's Hospital of Nanjing Medical University, from 2020 to 2021. Laboratory test results of these two children revealed hyponatremia (< 135.00 mmol/L), hyperkalemia (> 5.50 mmol/L), and hyperaldosteronism (> 180.00 ng/L). Moreover, genetic tests demonstrated no genetic variants highly associated with the phenotype in both cases. The two patients were subsequently treated with electrolyte correction. One of them also treated with antibiotics and one of them underwent surgery. They were followed for 8 and 4 months, respectively. No complications were observed during the follow-up period. This review aimed to outline both cases with parental consent. Conclusion: Transient pseudohypoaldosteronism should be considered in children younger than 6 months, presenting with vomiting, poor appetite, unexplained hyponatremia, hyperkalemia, elevated aldosterone levels, and urethral malformation or urinary tract infection. Furthermore, attention should be paid to whether salt supplementation or anti-infection therapy is effective.

Amino acid catabolism regulates hematopoietic stem cell proteostasis via a GCN2-eIF2α axis.

Hematopoietic stem cells (HSCs) adapt their metabolism to maintenance and proliferation; however, the mechanism remains incompletely understood. Here, we demonstrated that homeostatic HSCs exhibited high amino acid (AA) catabolism to reduce cellular AA levels, which activated the GCN2-eIF2α axis, a protein synthesis inhibitory checkpoint to restrain protein synthesis for maintenance. Furthermore, upon proliferation conditions, HSCs enhanced mitochondrial oxidative phosphorylation (OXPHOS) for higher energy production but decreased AA catabolism to accumulate cellular AAs, which inactivated the GCN2-eIF2α axis to increase protein synthesis and coupled with proteotoxic stress. Importantly, GCN2 deletion impaired HSC function in repopulation and regeneration. Mechanistically, GCN2 maintained proteostasis and inhibited Src-mediated AKT activation to repress mitochondrial OXPHOS in HSCs. Moreover, the glycolytic metabolite, NAD+ precursor nicotinamide riboside (NR), accelerated AA catabolism to activate GCN2 and sustain the long-term function of HSCs. Overall, our study uncovered direct links between metabolic alterations and translation control in HSCs during homeostasis and proliferation.

Antisense oligonucleotides rescue an intronic splicing variant in the ABCB11 gene that causes progressive familial intrahepatic cholestasis type 2.

BACKGROUND: Progressive familial intrahepatic cholestasis type 2 (PFIC2) is a rare disorder caused by variants in the ABCB11 gene encoding the bile salt export pump (BSEP). We investigated the molecular defect in a PFIC2 infant and rescued the splicing defect with antisense oligonucleotides (ASOs). METHODS: Whole-exome sequencing (WES) revealed compound heterozygous variants in the ABCB11 gene in a PFIC2 patient. Liver biopsy was immunostained for BSEP. The splicing effect of the candidate variants was investigated by minigene assay. ASOs were designed to rescue aberrant splicing. RESULTS: A Chinese girl of two nonconsanguineous healthy parents suffered from low glutamyl transpeptidase cholestasis and showed no response to the ursodeoxycholic acid. WES revealed that the patient was compound heterozygous for two novel variants in the ABCB11 gene: c.76+29T>G and c.390-2A>G. Liver immunohistochemistry showed the absence of BSEP. The variant c.76+29T>G was confirmed to retain 42 bp in the mature mRNA. The variant c.390-2A>G was confirmed to cause exon 6 skipping. We designed two ASOs and identified one of them that efficiently induced pseudoexon exclusion. CONCLUSION: We reported two novel variants of the ABCB11 gene, c.76+29T>G and c.390-2A>G, in a PFIC2 infant, thereby expanding the genotype of PFIC2. Our findings provide evidence for ASOs as a therapeutic approach for PFIC2 patients carrying intronic variants.

[Genetic analysis of PYGL gene variants for a child with Glycogen storage disease VI].

OBJECTIVE: To explore the clinical features and genetic basis of a patient with glycogen storage disease type VI (GSD-VI). METHODS: Clinical data of the patient was collected. Genomic DNA was extracted from peripheral blood samples of the proband and his parents. Genetic variants were detected by using whole exome sequencing. Candidate variants were verified by Sanger sequencing followed by bioinformatics analysis. RESULTS: The proband presented fasting hypoglycemia, hepatomegaly, growth retardation, transaminitis, metabolic acidosis and hyperlactatemia. Liver biopsy indicated GSD. Novel compound heterozygous PYGL gene variants (c.2089A>G/c.158_160delACT) were detected in the proband. Compound heterozygosity was confirmed by Sanger sequencing of the patient's genomic DNA. Provean and MutationTaster predicted the two variants as deleterious and the variant sites are highly conserved. CONCLUSION: The compound heterozygous variants (c.2089A>G/c.158_160delACT) of PYGL gene probably underlay the GSD in the patient. The two novel variants have expanded the spectrum of PYGL gene variants and provided the basis for genetic counseling of the family.

Retrospective analysis of pediatric patients with multiple rare-earth magnets ingestion: a single-center experience from China.

BACKGROUND: The aim of this study was to characterize patients who ingested multiple rare-earth magnets, reveal the harm of rare-earth magnet foreign bodies in the digestive tract, and develop a clinical management algorithm. METHODS: This was a retrospective review of patients with rare-earth magnet foreign bodies in the digestive tract admitted to a university-affiliated pediatric medical center in China, between January 2016 and December 2019; the subset of medical data evaluated included clinical symptoms, signs, treatments and outcomes. RESULTS: A total of 51 cases were included in this study, including 36(70.6%) males and 15(29.4%) females. The magnets were passed naturally in 24(47.1%) patients and removed by intervention in 27(52.9%) patients, including 5(9.8%) cases by endoscopy and 22(43.1%) cases by surgery. Twenty-two (43.1%)cases had gastrointestinal obstruction, perforation, and fistula. Compared with the non-surgical group, the time of the surgical group from ingestion to arriving at the hospital was longer([80(5-336) vs 26(2-216)]hours, p < 0.001) while there was no significant difference in the mean age or the number of magnets swallowed. CONCLUSIONS: Magnets are attractive to children, but lead to catastrophic consequences including gastrointestinal obstruction, perforation, and surgical interventions when ingested multiple magnets. Endoscopic resection should be urgently performed in the presence of multiple magnets as early as possible within 24 h, even in asymptomatic patients.

[A single-center retrospective analysis of 46 children with aerophagia].

OBJECTIVE: To study the clinical features of aerophagia in children. MEYJODS: A retrospective analysis was performed on the medical data of 46 children with aerophagia who were diagnosed and treated in Children's Hospital Affiliated to Nanjing Medical University from October 2011 to September 2019. RESULTS: Among these 46 children, 15 (33%) had Tourette syndrome. Abdominal distension was the most common symptom and was observed in 45 children (98%). The 24-hour esophageal multichannel intraluminal impedance monitoring showed a mean number of 341 times of air swallowing and a mean number of 212 times of gas reflux, and 95% of gas refluxes occurred in the upright body position. Compared with those without Tourette syndrome, the children with Tourette syndrome had a significantly higher incidence rate of air swallowing symptoms (67% vs 6%, P<0.001), but there were no significant differences in other symptoms and the results of 24-hour esophageal impedance. Dietary adjustment, psycho-behavioral therapy, and drug intervention significantly improved the scores of clinical symptoms and quality of life, among which psycho-behavioral therapy was an important intervention measure. CONCLUSIONS: Some children with aerophagia may have Tourette syndrome, and such children are more likely to have air swallowing symptoms. Psycho-behavioral therapy is one of the most important treatment methods, and children with aerophagia tend to have a good prognosis after treatment.

Analysis of STK11 gene variant in five Chinese patients with Peutz-Jeghers syndrome.

BACKGROUND: Peutz-Jeghers syndrome (PJS) is a rare autosomal dominant inherited disorder characterized by gastrointestinal hamartomatous polyps and mucocutaneous pigmentation. Germline mutation of a serine/threonine kinase 11(STK11) gene has been identified as a cause of PJS. In this study, we investigated the molecular basis of five Chinese PJS patients. METHODS: Blood samples were collected from five unrelated Chinese PJS patients and their parents. The entire coding region of the STK11 gene was amplified by polymerase chain reaction and analyzed by direct sequencing. RESULTS: Three different frameshift mutations (c.519insTGTG, c.792_793insT, and c.334_335insC), all of which would cause truncation of the gene product, were found in three patients. One missense mutation (p.Ser307Thr) and one 3bp deletion mutation (c.228-230del CGT) were identified in the remaining two patients. All of the five investigated patients carried de novo mutations. CONCLUSIONS: The results support that mutation of the LKB1 gene is a cause of PJS, and expand the spectrum of the STK11 gene mutations.