Living donor liver transplantation for myocerebrohepatopathy spectrum due to POLG mutations.

BACKGROUND: POLG is one of several nuclear genes associated with mitochondrial DNA maintenance defects and is a group of diseases caused by mitochondrial DNA deficiency that results in impaired adenosine triphosphate production and organ dysfunction. Myocerebrohepatopathy spectrum (MCHS) is the most severe and earliest presentation of POLG mutations, and liver transplantation (LT) for MCHS has never been reported. CASE PRESENTATION: The patient was a 3-month-old boy with acute liver failure and no neurological manifestations (e.g., seizures). We performed a living donor LT using a left lateral segment graft from his father. The postoperative course was uneventful. Subsequently, a homozygous POLG mutation (c.2890C>T, p. R964C) was identified by multigene analysis of neonatal/infantile intrahepatic cholestasis. Moreover, respiratory chain complex I, II, and III enzyme activities and the ratio of mtDNA to nuclear DNA in the liver were reduced. Therefore, we considered that these clinical manifestations and examination findings met the definition for MCHS. During meticulous follow-up, the patient had shown satisfactory physical growth and mental development until the time of writing this report. CONCLUSION: We presumed that the absence of remarkable neurologic manifestations prior to LT in patients with MCHS is a good indication for LT and contributes to a better prognosis in the present case.

Recurrent tandem duplication of UNC13D in familial hemophagocytic lymphohistiocytosis type 3.

Familial hemophagocytic lymphohistiocytosis type 3 is a fatal inborn error of immunity due to abnormal cytotoxic activity of T and NK cells and is caused by variants in UNC13D, which encodes Munc13-4. One published case was reported to carry a tandem duplication of UNC13D exons 7-12, and we here present another case with the exact same duplication breakpoints. The patient carried the tandem duplication from maternal origin, and a c.2346_2349 variant on the paternal allele. Single nucleotide polymorphism analysis around UNC13D revealed that the allele with tandem duplication was most likely a founder allele. Transposable element analysis showed that the breakpoints occurred within Alu elements in introns 12 and 6. Multiple sequence alignment revealed that Alu elements containing the truncated points are highly homologous. Sequence homology was thought to be a factor predisposing to the tandem duplication variant.

Rapid Flow Cytometry-Based Assay for the Functional Classification of MEFV Variants.

PURPOSE: Pathogenic MEFV variants cause pyrin-associated autoinflammatory diseases (PAADs), which include familial Mediterranean fever (FMF), FMF-like disease, and pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). The diagnosis of PAADs is established by clinical phenotypic and genetic analyses. However, the pathogenicity of most MEFV variants remains controversial, as they have not been functionally evaluated. This study aimed to establish and validate a new functional assay to evaluate the pathogenicity of MEFV variants. METHODS: We transfected THP-1 monocytes with 32 MEFV variants and analyzed their effects on cell death with or without stimulation with Clostridium difficile toxin A (TcdA) or UCN-01. These variants were classified using hierarchical cluster analysis. Macrophages were obtained from three healthy controls and two patients with a novel homozygous MEFVP257L variant, for comparison of IL-1ß secretion using a cell-based assay and a novel THP-1-based assay. RESULTS: Disease-associated MEFV variants induced variable degrees of spontaneous or TcdA/UCN-01-induced cell death in THP-1. Cell death was caspase-1 dependent and was accompanied by ASC speck formation and IL-1ß secretion, indicating that pathogenic MEFV variants induced abnormal pyrin inflammasome activation and subsequent pyroptotic cell deaths in this assay. The MEFV variants (n = 32) exhibiting distinct response signatures were classified into 6 clusters, which showed a good correlation with the clinical phenotypes. Regarding the pathogenicity of MEFVP257L variants, the results were consistent between the cell-based assay and the THP-1-based assay. CONCLUSION: Our assay facilitates a rapid and comprehensive assessment of the pathogenicity of MEFV variants and contributes to a refined definition of PAAD subtypes.

Immunophenotyping of A20 haploinsufficiency by multicolor flow cytometry.

Haploinsufficiency of A20 (HA20) causes inflammatory disease resembling Behçet's disease; many cases have been reported, including some that are complicated with autoimmune diseases. This study aims to clarify the immunophenotype of patients with HA20 by analyzing lymphocyte subsets using multicolor flow cytometry. The patients with HA20 previously diagnosed in a nationwide survey were compared by their cell subpopulations. In total, 27 parameters including regulatory T cells (Tregs), double-negative T cells (DNTs), and follicular helper T cells (TFHs) were analyzed and compared with the reference values in four age groups: 0-1, 2-6, 7-19, and ≥20 years. The Tregs of patients with HA20 tended to increase in tandem with age-matched controls at all ages. In addition, patients ≥20 years had increased DNTs compared with controls, whereas TFHs significantly increased in younger patients. In HA20 patients, the increase in DNTs and TFHs may contribute to the development of autoimmune diseases.

Human CTL-based functional analysis shows the reliability of a munc13-4 protein expression assay for FHL3 diagnosis.

Familial hemophagocytic lymphohistiocytosis (FHL) is the major form of hereditary hemophagocytic lymphohistiocytosis (HLH); as such, it requires prompt and accurate diagnosis. We previously reported that FHL type 3 (FHL3) can be rapidly screened by detecting munc13-4 expression in platelets using flow cytometry; however, the reliability of the munc13-4 expression assay for FHL3 diagnosis is unclear. Regardless of the type of UNC13D mutation, all reported FHL3 cases examined for the munc13-4 protein showed significantly reduced expression. However, the translated munc13-4 protein of some reportedly disease-causing UNC13D missense variants has not been assessed in terms of expression or function; therefore, their clinical significance remains unclear. The aim of this study was to determine the reliability of a munc13-4 expression assay for screening FHL3. Between 2011 and 2016, 108 HLH patients were screened by this method in our laboratory, and all 15 FHL3 patients were diagnosed accurately. To further elucidate whether munc13-4 expression analysis can reliably identify FHL3 patients harboring missense mutations in UNC13D, we developed an alloantigen-specific cytotoxic T lymphocyte (CTL) line and a CTL line immortalized by Herpesvirus saimiri derived from FHL3 patients. We then performed a comprehensive functional analysis of UNC13D variants. Transient expression of UNC13D complementary DNA constructs in these cell lines enabled us to determine the pathogenicity of the reported UNC13D missense variants according to expression levels of their translated munc13-4 proteins. Taken together with previous findings, the results presented herein show that the munc13-4 protein expression assay is a reliable tool for FHL3 screening.

Characterization of a large UNC13D gene duplication in a patient with familial hemophagocytic lymphohistiocytosis type 3.

Familial hemophagocytic lymphohistiocytosis (FHL) type 3 is a life-threatening immune dysregulation syndrome caused by mutations in the UNC13D gene, encoding the munc13-4 protein, which is important for function of cytotoxic lymphocytes. FHL3 accounts for 30-40% of FHL cases, and more than 100 mutations in the UNC13D gene have been described to date. We describe the first case of FHL3 carrying an intragenic duplication of UNC13D, apparently mediated by recombination of Alu elements. NK cell degranulation and munc13-4 protein expression assays are useful for early identification of such mutations, which may be missed by analysis of genomic DNA alone.

A CD57+ CTL Degranulation Assay Effectively Identifies Familial Hemophagocytic Lymphohistiocytosis Type 3 Patients.

PURPOSE: Familial hemophagocytic lymphohistiocytosis type 3 (FHL3) is a genetic disorder that results in immune dysregulation. It requires prompt and accurate diagnosis. A natural killer (NK) cell degranulation assay is often used to screen for FHL3 patients. However, we recently encountered two cases of late-onset FHL3 carrying novel UNC13D missense mutations: in these cases, the degranulation assays using freshly isolated and interleukin (IL)-2-activated NK cells yielded contradictory results. Since the defective degranulation of CD57+ cytotoxic T lymphocytes (CTLs) in these cases was helpful for making the diagnosis, we assessed whether the CD57+ CTL degranulation assay more effectively identified FHL3 patients than the NK cell assays. METHODS: Forty additional patients with hemophagocytic lymphohistiocytosis were prospectively screened for FHL3 by measuring the perforin expression in NK cells and the expression of Munc13-4, syntaxin-11, and Munc18-2 in platelets and by performing NK cell and CTL degranulation assays. The results were confirmed by genetic analysis. RESULTS: The freshly isolated NK cell degranulation assay detected FHL3 patients with high sensitivity (100%) but low specificity (71%). The IL-2-stimulated NK cell assay had improved specificity, but 3 out of the 31 non-FHL3 patients still showed degranulation below the threshold level. The CD57+ CTL degranulation assay identified FHL3 patients with high sensitivity and specificity (both 100%). CONCLUSIONS: The CD57+ CTL degranulation assay more effectively identified FHL3 patients than the NK cell-based assays.

Diagnostic accuracy of endoscopic features of pediatric acute gastrointestinal graft-versus-host disease.

BACKGROUND AND AIM: Acute gastrointestinal graft-versus-host disease (GI-GVHD) is a major cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT). There are very few studies on specific endoscopic findings in pediatric acute GI-GVHD. The aim of this retrospective case-control study was to elucidate the characteristic endoscopic findings in pediatric acute GI-GVHD that improve the diagnostic accuracy of endoscopy. METHODS: All consecutive patients under 18 years of age who underwent allogeneic HSCT in Kyoto University Hospital from May 2003 to October 2014 were identified retrospectively. Patients who underwent GI endoscopy as a result of sustained GI symptoms were identified. Intestinal villous patterns were evaluated by magnification endoscopy with the water-immersion technique. The patients were diagnosed with acute GI-GVHD and non-GVHD on the basis of biopsy histology. Endoscopic findings of the two groups were compared. RESULTS: Of the 171 patients who underwent HSCT, 30 underwent GI endoscopy. Of these, 17 and nine were diagnosed with acute GI-GVHD and non-GVHD, respectively. Compared with non-GVHD, acute GI-GVHD was associated significantly more often with short blunt villi in the duodenum (P = 0.013), variable defect villi and short blunt villi in the ileum (P = 0.009 and 0.035, respectively), and edema, erosion, and tortoiseshell-like mucosae in the colon (P = 0.017, 0.023, and 0.017, respectively). CONCLUSION: Pediatric acute GI-GVHD was associated with several characteristic features on magnifying endoscopy with the water-immersion technique. These features will be useful for endoscopic diagnosis of pediatric acute GI-GVHD.

Laboratory parameters identify familial haemophagocytic lymphohistiocytosis from other forms of paediatric haemophagocytosis.

Haemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome of immune dysregulation and is classified as primary or secondary according to the underlying aetiology. The treatment strategies recommended for these two groups differ substantially; however, it is thought to be impossible to predict the underlying causes of HLH using conventional laboratory tests. Recent studies show that serum levels of soluble interleukin-2 receptor (sIL2R) and ferritin are useful for differentiating some forms of HLH. The present study reports that combinations of common laboratory parameters, such as the percentage of total lymphocytes within the peripheral blood leucocyte population, serum levels of lactate dehydrogenase and the sIL2R/ferritin ratio, are useful for identifying patients with familial haemophagocytic lymphohistiocytosis and for differentiating the underlying aetiology of paediatric HLH during the early course of the disease. These findings suggest that the pathogenesis of HLH differs greatly in terms of innate and adaptive immunity depending on the aetiology and may provide a new approach to unravelling the complex pathophysiology underlying this syndrome.

Acute lymphoblastic leukemia in a girl with Wilson's disease.

Wilson's disease (WD) is an autosomal recessive defect in cellular copper transportation. Although acute lymphoblastic leukemia (ALL) is the most common form of childhood malignancy, only two cases of ALL associated with WD have been reported to date. One patient died of relapse and infection, and the other died of neutropenic sepsis during the treatment. We here describe the case of a 10-year-old girl with WD and ALL. Adverse events of chemotherapy, including liver toxicity and severe myelosuppression, necessitated adjustments in the chemotherapy doses. After completion of the treatment, the patient has remained in remission from ALL without progression of liver damage for 2 years. Severe treatment-related toxicity should be considered in chemotherapy for patients with WD.

Sustained high plasma mannose less sensitive to fluctuating blood glucose in glycogen storage disease type Ia children.

Plasma mannose is suggested to be largely generated from liver glycogen-oriented glucose-6-phosphate. This study examined plasma mannose in glycogen storage disease type Ia (GSD Ia) lacking conversion of glucose-6-phosphate to glucose in the liver. We initially examined fasting--and postprandial 2 h--plasma mannose and other blood carbohydrates and lipids for seven GSD Ia children receiving dietary interventions using cornstarch and six healthy age-matched children. Next, one-day successive intra-individual parameter changes were examined for six affected and two control children. Although there were no significant differences in fasting--and postprandial 2 h--glucose and insulin levels, the mannose level of the affected group was invariably much higher than that of the control group (p < 0.001): the fasting level of the affected group was about two-fold that of the control group; the postprandial-2 h level remained almost unchanged in the affected group, although it was one-half of the fasting level in the control group. Inter-individual analyses revealed that the GSD Ia group mannose level was significantly and positively correlated with lactate and triglycerides levels at both time points (p < 0.01). In each control, mannose levels fluctuated greatly, maintaining strong and significant negative correlations with glucose and insulin levels (p < 0.001). Correlations were lower or nonexistent in GSD Ia children. In individuals with high lactate and triglycerides levels, strikingly high mannose levels never changed against glucose and insulin fluctuations. Plasma mannose is less sensitive to blood glucose and insulin in GSD Ia children. Its basal level and the fluctuation pattern differ by their metabolic activity.

[Utility of endoscopic ultrasound-guided fine needle aspiration for diagnosis of Burkitt lymphoma in two pediatric cases].

Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) was performed as a diagnostic procedure for two pediatric patients with intra-abdominal tumors. Case 1 was an 8-year-old boy with a huge tumor in the portal-hepatic region. Case 2 was a 17-year-old girl with a history of diabetes and recurrent relapse of Burkitt lymphoma, who had a newly developing tumor in the pancreatic body. In both cases, EUS-FNA was performed as a less invasive diagnostic procedure than open biopsy or total resection of the tumor. Tumor cells were determined to be of the B cell lineage by flow cytometric and immunostaining analyses. Both cases were diagnosed as having Burkitt lymphoma based on detection of IgH/C-MYC translocation by FISH. The aspiration was successfully conducted without severe complications, and both patients were immediately given chemotherapy. EUS-FNA is a safe and minimally invasive procedure with high diagnostic value for pediatric cases with intra-abdominal tumors.

Clinical outcome of ulcerative colitis with severe onset in children: a multicenter prospective cohort study.

BACKGROUND: As best practices for treating children with severe-onset ulcerative colitis remain controversial in the era of biologic agents, we prospectively investigated treatments and outcomes in a multicenter cohort. METHODS: Using a Web-based data registry maintained in Japan between October 2012 and March 2020, we compared management and treatment outcomes in an S1 group defined by a Pediatric Ulcerative Colitis Activity Index of 65 or more points at diagnosis with those in an S0 group defined by an index value below 65. RESULTS: Three hundred one children with ulcerative colitis treated at 21 institutions were included, with follow-up for 3.6 ± 1.9 years. Among them, 75 (25.0%) were in S1; their age at diagnosis was 12.3 ± 2.9 years, and 93% had pancolitis. Colectomy free rates in S1 were 89% after 1 year, 79% after 2, and 74% after 5, significantly lower than for S0 (P = 0.0003). Calcineurin inhibitors and biologic agents, respectively, were given to 53% and 56% of S1 patients, significantly more than for S0 patients (P < 0.0001). Among S1 patients treated with calcineurin inhibitors when steroids failed, 23% required neither biologic agents nor colectomy, similarly to the S0 group (P = 0.46). CONCLUSIONS: Children with severe ulcerative colitis are likely to require powerful agents such as calcineurin inhibitors and biologic agents; sometimes colectomy ultimately proves necessary. Need for biologic agents in steroid-resistant patients might be reduced to an extent by interposing a therapeutic trial of CI rather than turning to biologic agents or colectomy immediately.

Fatty liver and anti-oxidant enzyme activities along with peroxisome proliferator-activated receptors γ and α expressions in the liver of Wilson's disease.

BACKGROUND: The mechanisms of liver damage and steatosis in Wilson's disease (WD) presenting accumulation of copper generating oxidants remain unclear. Recent studies have shown that peroxisome proliferator-activated receptors (PPARs), in particular PPARs α and γ, regulate fat content of the liver together with the anti-oxidant and anti-inflammation systems. However, such PPARs have never been studied in WD. METHODS: We examined PPARs along with the liver damage and steatosis of WD using liver specimens from affected patients exhibiting mild liver damage (group I, n = 5), moderate or greater liver damage (group II, n = 10) and fulminant hepatic failure (group III, n = 5), and from asymptomatic carriers (group H, n = 4). RESULTS: PPAR α expression was increased over the control levels in groups H and I but was decreased in groups II and III in parallel with the progression of liver damage (group H = I>II>III). PPAR γ expression was inversely increased (group H

Acute liver failure in young children with systemic-onset juvenile idiopathic arthritis without macrophage activation syndrome: report of two cases.

Acute liver failure (ALF) with macrophage activation syndrome (MAS) is well known as a complication of systemic-onset juvenile idiopathic arthritis (S-JIA). However, liver failure without overt MAS is rare in S-JIA. We encountered two Japanese children with S-JIA in whom ALF developed during the remission of clinical manifestations. ALF without MAS was improved with plasma exchange and cyclosporine A combined with pulse methylprednisolone.

Case Report: A Pediatric Case of Familial Mediterranean Fever Concurrent With Autoimmune Hepatitis.

Familial Mediterranean fever (FMF) is a hereditary, autoinflammatory disease that causes recurrent fever, arthritis, and serositis. The diagnosis of FMF is based on the presentation of typical clinical symptoms and the Mediterranean fever gene (MEFV) test. However, the challenge lies in diagnosing atypical cases. In this report, we have described a pediatric patient with complex FMF whose diagnosis required trio-whole exome sequencing (WES) and functional validation of a rare MEFV variant. A 3-year-old boy presented with recurrent episodes of elevated liver enzymes and arthralgia. He was diagnosed with autoimmune hepatitis (AIH), and his liver enzymes improved rapidly with steroid treatment. However, he exhibited recurrent arthralgia and severe abdominal attacks. Trio-WES identified compound heterozygous mutations in MEFV (V726A and I692del). Ex vivo functional assays of the patient's monocytes and macrophages, which had been pre-treated with Clostridium difficile toxin A (TcdA) and colchicine, were comparable to those of typical FMF patients, thereby confirming the diagnosis of FMF. Although he was intolerant to colchicine because of liver toxicity, subsequent administration of canakinumab successfully ameliorated his abdominal attacks. However, it was ineffective against liver injury, which recurred after steroid tapering. Therefore, in this case, the pathogenesis of AIH was probably interleukin-1ß (IL-1ß)-independent. In fact, AIH might have been a concurrent disease with FMF, rather than being one of its complications. Nevertheless, further studies are necessary to determine whether FMF-induced inflammasome activation contributes to AIH development. Moreover, we must consider the possibility of mixed phenotypes in such atypical patients who present distinct pathologies simultaneously.