Novel insights into presenilin 1 mutation associated with a distinctive dementia phenotype and cotton wool plaques.

BACKGROUND: The mutations in the presenilin 1 gene (PSEN1) are the main cause of familial Alzheimer's disease. PSEN1 mutations affect amyloid-beta peptide production, which accumulates in the brain as senile plaque and cotton wool plaques (CWPs) and relates to other neurodegenerative disorders. Here we report the second case of the PSEN1 G266S mutation, which showed distinctive neuropathological features, including abundant CWPs. Lewy body pathology, and altered amyloid-beta production. METHOD: Using the proband's samples, we performed genetic analysis of the PSEN1, APP, MAPT, and APOE genes, histopathological and immunohistochemical analysis of the brain tissue, and biochemical analysis of Aß production in COS cells transfected with wild-type or mutant PSEN1. RESULTS: The patient presented with memory loss, abnormal behavior, and visual hallucinations. Brain scans showed reduced blood flow, mild atrophy, and white matter lesions. Genetic analysis revealed a heterozygous mutation at codon 266 (G266S) of PSEN1 and polymorphism of MAPT (Q230R). The brain had many CWPs, severe cerebral amyloid angiopathy (CAA), senile plaque, Lewy bodies, and neurites. Electron microscopy displayed myelinated fiber degeneration, mitochondrial damage, and amyloid fibrils in the white matter. The production level of Aß42 in PSEN1 G266S-transfected cells significantly increased. CONCLUSION: Our findings suggest that the PSEN1 G266S mutation may cause a heterogeneous clinical and pathological phenotype, influenced by other genetic or environmental factors.

Comprehensive analyses of neurodevelopmental outcomes and quality of life of children with biliary atresia.

OBJECTIVES: To holistically evaluate neurodevelopmental outcomes and quality of life (QOL) of Japanese patients with biliary atresia (BA) and to investigate the factors associated with the outcomes. METHODS: This study enrolled patients with BA aged 5-18 years who visited Osaka University Hospital in 2021. Neurodevelopmental assessments were performed to evaluate intellectual ability, cognitive functions and adaptive skill levels. Furthermore, emotional and behavioral issues, characteristics of attention deficit hyperactivity disorder, and QOL were concomitantly assessed in the same cohort. Biochemical and social factors associated with the results were examined. RESULTS: Fifty-three patients, with a median age of 11.2 years were included in the analyses. Patients with BA had a significantly lower Full-Scale Intelligence Quotient or developmental quotient (FSIQ/DQ) score and Vineland Adaptive Behavior Scale (VABS) composite score than the general Japanese population. Household education level and short stature were associated with low and borderline FSIQ/DQ and VABS composite scores, respectively. Among patients with low and borderline FSIQ/DQ scores, those with average or high VABS composite scores received significantly less neuroeducational care than those with low and borderline VABS composite scores. Despite the low FSIQ/DQ and VABS composite scores, the total QOL scores were higher than those of the general population. CONCLUSION: Patients with BA had intellectual and behavioral impairments. Notably, patients with intellectual impairments are overlooked and not followed up, especially if adaptive skills are maintained.

Rapid Chemical Synthesis of Serine Protease Inhibitor Kazal-type 13 (SPINK13) Glycoform by a Combined Method with Glycan Insertion Strategy and Fast-Flow Fmoc SPPS.

Serine protease inhibitor Kazal type 13 (SPINK13) is a secreted protein that has been recently studied as a therapeutic drug and an interesting biomarker for cancer cells. Although SPINK13 has a consensus sequence (Pro-Asn-Val-Thr) for N-glycosylation, the existence of N-glycosylation and its functions are still unclear. In addition to this, the preparation of glycosylated SPINK 13 has not been examined by both the cell expression method and chemical synthesis. Herein we report the chemical synthesis of the scarce N-glycosylated form of SPINK13 by a rapid synthetic method combined with the chemical glycan insertion strategy and a fast-flow SPPS method. Glycosylated asparagine thioacid was designed to chemoselectively be inserted between two peptide segments where is the sterically bulky Pro-Asn(N-glycan)-Val junction by two coupling reactions which consist of diacyl disulfide coupling (DDC) and thioacid capture ligation (TCL). This insertion strategy successfully afforded the full-length polypeptide of SPINK13 within two steps from glycosylated asparagine thioacid. Because the two peptides used for this synthesis were prepared by a fast-flow SPPS, the total synthetic time of glycoprotein was considerably shortened. This synthetic concept enables us to repetitively synthesize a target glycoprotein easily. Folding experiments afforded well-folded structure confirmed by CD and disulfide bond map. Invasion assays of glycosylated SPINK13 and non-glycosylated SPINK13 with pancreatic cancer cells showed that non-glycosylated SPINK-13 was more potent than that of glycosylated SPINK13.

Twin research shows glycan changes are more susceptible to environmental factors than their carrier glycoproteins.

Changes in protein glycosylation are clinically used as biomarkers. In the present study, we employed a twin cohort to investigate the contributions of genetic and environmental factors to glycan modifications of glycoproteins. Mac-2 binding protein (Mac-2 bp), haptoglobin (Hp), and their glycosylated forms are liver fibrosis and cancer biomarkers. Sera from 107 twin pairs without clinical information were used as a training cohort for the Mac-2 bp and Mac-2 bp glycosylation isomer (M2BPGi) assay. As a validation cohort, 22 twin pairs were enrolled in the study. For each twin pair, one twin was diagnosed with liver or pancreatic disease. For the training cohort, the correlation ratios of serum Mac-2 bp and M2BPGi levels in twin sera with random sequences were 0.30 and 0.018, respectively. The correlation ratios between twin pairs in the validation cohort for serum Mac-2 bp and M2BPGi levels were 0.75 and 0.35, respectively. In contrast, correlation ratios of serum Hp and fucosylated haptoglobin (Fuc-Hp) levels between twin sera with liver and pancreatic disease were 0.49 and 0.16, respectively. Although serum protein levels of glycoproteins are susceptible to genetic factors, characteristic glycan changes of these glycoproteins are more susceptible to environmental factors, including liver and pancreatic disease.

The impact of treatment on the psychological burden of mothers of children with chronic hepatitis C virus infection: a multicenter, questionnaire survey.

Mothers of children with chronic hepatitis C virus (HCV) infection experience anxiety about the health of their children. In this study we assessed an impact of treating children with chronic HCV infection on the psychological burden of their mothers. This was a multicenter, questionnaire survey conducted at six institutions in Japan. A newly-developed questionnaire for this study was used to assess changes in the mothers' various concerns regarding HCV infection and thoughts about their child's HCV infection. Responses at the time of diagnosis and at the time of the survey were compared between mothers of children who had received treatment and those without treatment. Responses were received from 36 of 37 eligible mothers (11 and 25, non-treatment and treatment groups, respectively). All children in treatment group had successfully eliminated the virus. Mothers in both groups were psychologically stressed in various ways, including concern about their child's health in the present and future at the time of diagnosis, concern about school, employment, and marriage, concern about the behavior of others towards them and infecting others with HCV, and feelings of guilt regarding their child. These concerns were significantly lower in the present compared to at the time of diagnosis in treatment group, and the rate of decrease was significantly higher in treatment group compared to non-treatment group. Successful treatment greatly reduced mothers' concerns about their children's HCV infection, indicating that treatment during childhood is beneficial from the perspective of the mothers' psychological burden.

Lewis glycosphingolipids as critical determinants of TRAIL sensitivity in cancer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cancer cell death and contributes to tumor rejection by cytotoxic lymphocytes in cancer immunosurveillance and immunotherapy. TRAIL and TRAIL receptor agonists have garnered wide popularity as promising agents for cancer therapy. We previously demonstrated that the loss of fucosylation in cancer cells impairs TRAIL sensitivity; however, the precise structures of the fucosylated glycans that regulate TRAIL sensitivity and their carrier molecules remain elusive. Herein, we observed that Lewis glycans among various fucosylated glycans positively regulate TRAIL-induced cell death. Specifically, Lewis glycans on lacto/neolacto glycosphingolipids, but not glycoproteins including TRAIL receptors, enhanced TRAIL-induced formation of the cytosolic caspase 8 complex, without affecting the formation of the membranous receptor complex. Furthermore, type I Lewis glycan expression in colon cancer cell lines and patient-derived cancer organoids was positively correlated with TRAIL sensitivity. These findings provide novel insights into the regulatory mechanism of TRAIL-induced cell death and facilitate the identification of novel predictive biomarkers for TRAIL-related cancer therapies in future.

Novel gene mutations in three Japanese patients with ARC syndrome associated mild phenotypes: a case series.

BACKGROUND: Arthrogryposis, renal dysfunction, and cholestasis syndrome (ARCS) is a rare autosomal recessive disorder caused by mutations in VPS33B (ARCS1) and VIPAS39 (ARCS2). As per literature, most patients with ARCS died of persistent infections and bleeding by the age of 1 year. We report the first Japanese cases with ARCS1 and ARCS2 who presented with mild phenotypes and were diagnosed via genetic testing. CASE PRESENTATION: Case 1: A 6-year-old boy born to nonconsanguineous Japanese parents presented with jaundice and normal serum gamma-glutamyl transferase (GGT) levels, proteinuria, bilateral nerve deafness, motor delay, failure to thrive, and persistent pruritus. After cochlear implantation for deafness at the age of 2 years, despite a normal platelet count and prothrombin time-international normalized ratio, the patient presented with persistent bleeding that required hematoma removal. Although he did not show any obvious signs of arthrogryposis, he was suspected to have ARCS based on other symptoms. Compound heterozygous mutations in VPS33B were identified using targeted next-generation sequencing (NGS), which resulted in no protein expression. Case 2: A 7-month-old boy, the younger brother of case 1, presented with bilateral deafness, renal tubular dysfunction, failure to thrive, and mild cholestasis. He had the same mutations that were identified in his brother's VPS33B. Case 3: A 24-year-old man born to nonconsanguineous Japanese parents was suspected to have progressive familial intrahepatic cholestasis 1 (PFIC1) in his childhood on the basis of low GGT cholestasis, renal tubular dysfunction, sensory deafness, mental retardation, and persistent itching. A liver biopsy performed at the age of 16 years showed findings that were consistent with PFIC1. He developed anemia owing to intraperitoneal hemorrhage from a peripheral intrahepatic artery the day after the biopsy, and transcatheter arterial embolization was required. ARCS2 was diagnosed using targeted NGS, which identified novel compound heterozygous mutations in VIPAS39. CONCLUSIONS: The first Japanese cases of ARCS1 and ARCS2 diagnosed using genetic tests were reported in this study. These cases are milder than those previously reported. For patients with ARCS, invasive procedures should be performed with meticulous care to prevent bleeding.

Enterococcus spp. have higher fitness for survival, in a pH-dependent manner, in pancreatic juice among duodenal bacterial flora.

BACKGROUND AND AIM: Bacterial infection is involved in the progression of many gastrointestinal diseases, including those of pancreas; however, how and which bacteria colonize in pancreatic juice and tissue have yet to be elucidated. Recently, we reported that Enterococcus faecalis exists in the pancreatic juice and tissues of patients with chronic pancreatic disease. Here, we investigated the survival of E. faecalis in duodenal juice with different pH conditions. METHODS: Pancreatic juice samples from 62 patients with cancers of the duodeno-pancreato-biliary region were evaluated for the presence of E. faecalis. 16S ribosomal RNA polymerase chain reaction and 16S-based metagenome analyses were performed to determine the bacterial composition. The survival of E. faecalis in various pancreatic juice conditions was evaluated. RESULTS: Of 62 samples, 27% (17/62) were positive for Enterococcus spp., among which 71% (12/17) contained E. faecalis. Enterococcus spp. showed the highest fitness for survival in alkaline pancreatic juice among various bacterial species. The microbiome of pancreatic juice from patients with pancreatic and bile duct cancer showed diversity, but Enterococcus spp. were enriched among duodenal tumors and intraductal papillary mucinous neoplasms. CONCLUSIONS: Alkalinity is one of the important factors for the selective survival of E. faecalis among microbiota. E. faecalis can colonize the pancreatic duct when the pancreatic juice condition is altered.

Urinary iodine and thyroglobulin are useful markers in infants suspected of congenital hypothyroidism based on newborn screening.

OBJECTIVES: Iodine deficiency and excess both cause thyroid dysfunction. Few data describe the relationship between iodine status and outcomes of congenital hypothyroidism (CH) in iodine-sufficient areas. We investigated urinary iodine (UI) concentration and its relationship with the clinical course of CH. METHODS: We reviewed and retrospectively analyzed patients with positive newborn screening (NBS) for CH from January 2012 to June 2019 in Japan, obtaining UI and UI-urine creatinine ratio (UI/Cr), serum TSH, free T4, free T3 and thyroglobulin (Tg) at the first visit, TSH at NBS, levothyroxine (LT4) dose, and subsequent doses. A UI value of 100-299 µg/L was considered adequate. RESULTS: Forty-eight patients were included. Median UI and UI/Cr were 325 µg/L and 3,930 µg/gCr, respectively. UI was high (≥300 µg/L) in 26 (54%) and low (≤99 µg/L) in 11 (23%). LT4 was administered to 34 patients. Iodine status was not related to the need for treatment. We found a U-shaped relationship between Tg and UI/Cr. Patients with high Tg (≥400 ng/mL) and abnormal UI levels required significantly lower LT4 doses (≤20 µg/day) at three years of age. Even if they showed severe hypothyroidism initially, they did not need subsequent dose increments. CONCLUSIONS: Abnormal UI levels with Tg elevation were associated with lower LT4 dose requirements. The evaluation of iodine status and Tg concentrations were considered useful in patients suspected of CH.

Two girls with a neonatal screening-negative 21-hydroxylase deficiency requiring treatment with hydrocortisone for virilization in late childhood.

Herein, we report two girls with a neonatal screening (NS)-negative 21-hydroxylase deficiency (21-OHD) requiring treatment with hydrocortisone due to virilization that developed in late childhood. Patient 1 was born prematurely on the 30th gestational week with normal external genitalia at birth. She passed the NS for 21-OHD. At 6 yr of age, she was referred to a hospital for evaluation of premature pubarche and clitoromegaly. Her diagnosis was central precocious puberty, and GnRH agonist was initiated. However, her symptoms did not improve despite treatment for over 4 years. She was then referred to our hospital where she was diagnosed with 21-OHD. Although she was started on hydrocortisone therapy, her adult height reached only 140 cm (-3.4 SD). Patient 2 was delivered at 37 weeks of gestation and passed the NS for 21-OHD. She was referred to a hospital because of premature pubarche at the age of 6 yr. She was diagnosed with 21-OHD, and hydrocortisone replacement therapy was initiated. Her present height at 13 yr of age is 148 cm (-1.3 SD). These cases reminded us that the possibility of 21-OHD should be considered when patients show premature pubarche or precocious puberty, even if they passed the NS test for 21-OHD.

Matrix Gla protein maintains normal and malignant hematopoietic progenitor cells by interacting with bone morphogenetic protein-4.

Matrix Gla protein (MGP), a modulator of the BMP-SMAD signals, inhibits arterial calcification in a Glu γ-carboxylation dependent manner but the role of MGP highly expressed in a subset of bone marrow (BM) mesenchymal stem/stromal cells is unknown. Here we provide evidence that MGP might be a niche factor for both normal and malignant myelopoiesis. When mouse BM hematopoietic cells were cocultured with mitomycin C-treated BM stromal cells in the presence of anti-MGP antibody, growth of hematopoietic cells was reduced by half, and maintenance of long-term culture-initiating cells (LTC-ICs) was profoundly attenuated. Antibody-mediated blockage of MGP also inhibited growth (by a fifth) and cobblestone formation (by half) of stroma-dependent MB-1 myeloblastoma cells. MGP was undetectable in normal hematopoietic cells but was expressed in various mesenchymal cells and was aberrantly high in MB-1 cells. MGP and bone morphogenetic protein (BMP)-4 were co-induced in stromal cells cocultured with both normal hematopoietic cells and MB-1 myeloblastoma cells in an oscillating several days-periodic manner. BMP-2 was also induced in stromal cells cocultured with normal hematopoietic cells but was barely expressed when cocultured with MB-1 cells. GST-pulldown and luciferase reporter assays showed that uncarboxylated MGP interacted with BMP-4 and that anti-MGP antibody abolished this interaction. LDN-193189, a selective BMP signaling inhibitor, inhibited growth and cobblestone formation of MB-1 cells. The addition of warfarin, a selective inhibitor of vitamin K-dependent Glu γ-carboxylation, did not affect MB-1 cell growth, suggesting that uncarboxylated MGP has a biological effect in niche. These results indicate that MGP may maintain normal and malignant hematopoietic progenitor cells, possibly by modulating BMP signals independently of Glu γ-carboxylation. Aberrant MGP by leukemic cells and selective induction of BMP-4 relative to BMP-2 in stromal cells might specify malignant niche.

Translin restricts the growth of pubertal mammary epithelial cells estrogen-independently in mice.

Translin, a ubiquitous RNA/DNA-binding protein that forms a hetero-octamer together with Translin-associated factor X (TRAX), possesses endoribonuclease activity and plays a physiological role in restricting the size and differentiation of mesenchymal precursor cells. However, the precise role of Translin in epithelial cells remains unclear. Here, we show evidence that Translin restricts the growth of pubertal mammary epithelial cells. The mammary epithelia of Translin-null females exhibited retarded growth before puberty, but highly enhanced growth and DNA synthesis with increased ramification after the onset of puberty. Primary cultures of Translin-null mammary epithelial cells showed augmented DNA synthesis in a ligand-independent and ligand-enhanced manner. Translin-null ovariectomized mice implanted with slow-release estrogen pellets showed enhanced length and ramification of the mammary glands. Mammary epithelial growth was also observed in ovariectomized Translin-null mice implanted with placebo pellets. Luciferase reporter assays using embryonic fibroblasts from Translin-null mice showed unaltered estrogen receptor α function. These results indicate that Translin plays a physiological role in restricting intrinsic growth, beyond mesenchymal cells, of pubertal mammary epithelial cells.

PML-RARα induces all-trans retinoic acid-dependent transcriptional activation through interaction with MED1.

Transcriptional activation by PML-RARα, an acute promyelocytic leukemia-related oncofusion protein, requires pharmacological concentrations of all-trans retinoic acid (ATRA). However, the mechanism by which the liganded PML-RARα complex leads to the formation of the preinitiation complex has been unidentified. Here we demonstrate that the Mediator subunit MED1 plays an important role in the ATRA-dependent activation of the PML-RARα-bound promoter. Luciferase reporter assays showed that PML-RARα induced significant transcription at pharmacological doses (1 µM) of ATRA; however, this was submaximal and equivalent to the level of transcription driven by intact RARα at physiological doses (1 nM) of ATRA. Transcription depended upon the interaction of PML-RARα with the two LxxLL nuclear receptor recognition motifs of MED1, and LxxLL→LxxAA mutations led to minimal transcription. Mechanistically, MED1 interacted ATRA-dependently with the RARα portion of PML-RARα through the two LxxLL motifs of MED1. These results suggest that PML-RARα initiates ATRA-induced transcription through its interaction with MED1.

Possible involvement of Enterococcus infection in the pathogenesis of chronic pancreatitis and cancer.

(Aim) Bacterial infection underlies the pathogenesis of many human diseases, including acute and chronic inflammation. Here, we investigated a possible role for bacterial infection in the progression of chronic pancreatitis. (Materials and Methods) Pancreatic juice was obtained from patients with pancreatic cancer (n = 20) or duodenal cancer/bile duct cancer (n = 16) and subjected to PCR using universal primers for the bacterial 16S ribosomal RNA gene. Bacterial species were identified by PCR using bile samples from four pancreatic cancer patients. PCR products were subcloned into T-vectors, and the sequences were then analyzed. Immunohistochemical and serologic analyses for Enterococcus faecalis infection were performed on a large cohort of healthy volunteers and patients with chronic pancreatitis or pancreatic cancer and on mice with caerulein-induced chronic pancreatitis. The effect of E. faecalis antigens on cytokine secretion by pancreatic cancer cells was also investigated. (Results) We found that 29 of 36 pancreatic juice samples were positive for bacterial DNA. Enterococcus and Enterobacter species were detected primarily in bile, which is thought to be a pathway for bacterial infection of the pancreas. Enterococcus faecalis was also detected in pancreatic tissue from chronic pancreatitis and pancreatic cancer patients; antibodies to E. faecalis capsular polysaccharide were elevated in serum from chronic pancreatitis patients. Enterococcus-specific antibodies and pancreatic tissue-associated E. faecalis were detected in mice with caerulein-induced chronic pancreatitis. Addition of Enterococcus lipoteichoic acid and heat-killed bacteria induced expression of pro-fibrotic cytokines by pancreatic cancer cells in vitro. (Conclusion) Infection with E. faecalis may be involved in chronic pancreatitis progression, ultimately leading to development of pancreatic cancer.

Translin modulates mesenchymal cell proliferation and differentiation in mice.

Translin, a highly conserved DNA/RNA binding protein that forms a hetero-octamer together with Translin-associated factor X (TRAX), possesses a broad variety of functions, including RNA processing and DNA repair. Recent studies have reported that Translin is involved in mesenchymal cell physiology. Thus, here we analyzed the intrinsic role of Translin in mesenchymal cell proliferation and differentiation. Translin-deficient E11.5 mouse embryonic fibroblasts showed enhanced growth. Translin-deficient bone marrow-derived mesenchymal stem cells showed substantial expansion in vivo and enhanced proliferation in vitro. These cells also showed enhanced osteogenic and adipocytic differentiation. Histological analyses showed adipocytic hypertrophy in various adipose tissues. Translin knockout did not affect the growth of subcutaneous white adipose tissue-derived stem cells, but enhanced adipocytic differentiation was observed in vitro. Contrary to previous reports, in vitro-fertilized Translin-null mice were not runted and exhibited normal metabolic homeostasis, indicating the fragility of these mice to environmental conditions. Together, these data suggest that Translin plays an intrinsic role in restricting mesenchymal cell proliferation and differentiation.

Japanese clinical practice guidelines for allied disorders of Hirschsprung's disease, 2017.

BACKGROUND: Despite the presence of ganglion cells in the rectum, some patients have symptoms similar to those of Hirschsprung's disease. A consensus has yet to be established regarding the terminology for these diseases. We defined this group of diseases as "allied disorders of Hirschsprung's disease" and compiled these guidelines to facilitate accurate clinician diagnosis and provide appropriate treatment strategies for each disease. METHODS: These guidelines were developed using the methodologies in the Medical Information Network Distribution System (MINDS). Of seven allied disorders, isolated hypoganglionosis; megacystis-microcolon-intestinal hypoperistalsis syndrome; and chronic idiopathic intestinal pseudo-obstruction were selected as targets of clinical questions (CQ). In a comprehensive search of the Japanese- and English-language articles in PubMed and Ichu-Shi Web, 836 pieces of evidence related to the CQ were extracted from 288 articles; these pieces of evidence were summarized in an evidence table. RESULTS: We herein outline the newly established Japanese clinical practice guidelines for allied disorders of Hirschsprung's disease. Given that the target diseases are rare and intractable, most evidence was drawn from case reports and case series. In the CQ, the diagnosis, medication, nutritional support, surgical therapy, and prognosis for each disease are given. We emphasize the importance of full-thickness intestinal biopsy specimens for the histopathological evaluation of enteric ganglia. Considering the practicality of the guidelines, the recommendations for each CQ were created with protracted discussions among specialists. CONCLUSIONS: Clinical practice recommendations for allied disorders of Hirschprung's disease are given for each CQ, along with an assessment of the current evidence. We hope that the information will be helpful in daily practice and future studies.

Total Bile Acid Concentration in Duodenal Fluid Is a Useful Preoperative Screening Marker to Rule Out Biliary Atresia.

OBJECTIVES: Duodenal tube test (DTT) is used as a preoperative screening to rule out biliary atresia (BA). In previous reports, DTT was assessed by the color of the duodenal fluid, but there were no quantitative criteria. The aim of this study was to examine the efficacy of DTT based on the total bile acid (TBA) concentration in duodenal fluid. METHODS: This is a single-center retrospective study of infants with cholestasis who underwent DTT from 2008 to 2016 at the Osaka University Hospital. The cut-off values of maximum TBA in duodenal fluid (dTBA), dTBA/serum TBA ratio (sTBA), and dTBA/serum gamma-glutamyl transpeptidase (sGGT) ratio were assessed for the accuracy in excluding BA. RESULTS: A total of 37 infants were included in this study; 16 infants with BA and 21 infants with other causes of intrahepatic cholestasis. dTBA demonstrated sensitivity of 100% and specificity of 90.5% with the cut-off value of 16.8 µmol/L. Specificity was further improved to 95.2% with dTBA/sTBA ratio (cut-off value: 0.088) and 100% with dTBA/sGGT ratio (cut-off value: 0.076 µmol/U). DTT could be performed 0.8 ±â€Š1.4 days after admission. Hypoglycemia was developed in 1 infant. CONCLUSIONS: DTT evaluated by dTBA, dTBA/sTBA ratio, and dTBA/sGGT ratio had high accuracy to rule out BA and could avoid unnecessary surgery in some infants.