Intellectual disability and abnormal cortical neuron phenotypes in patients with Bloom syndrome.

Bloom syndrome (BS) is a rare autosomal recessive disorder characterized by genomic instability that leads to various complications, including cancer. Given the low prevalence of BS in Japan, we conducted a nationwide survey. We recruited eight patients with BS, three of whom exhibited intellectual disability. The 631delCAA mutation in the BLM gene was detected in 9 out of 16 alleles. To investigate neuronal development in patients with BS, we generated induced pluripotent stem cells derived from one of these patients (BS-iPSCs). We examined the phenotypes of the induced cortical neurons derived from the generated BS-iPSCs using a previously reported protocol; the generated BS-iPSCs showed an approximately 10-times higher frequency of sister-chromatid exchange (SCE) than the control iPSCs. Immunocytochemistry revealed shorter axons and higher proliferative potential in BS-iPSC-derived cortical neurons compared with control iPSCs. To our knowledge, our study is the first to clarify the abnormality of the cortical neuron phenotypes derived from patients with BS. Our findings may help identify the pathogenesis of neuronal differentiation in BS and aid in the development of novel therapeutic agents.

Rothmund-Thomson syndrome investigated by two nationwide surveys in Japan.

BACKGROUND: Rothmund-Thomson syndrome (RTS) is an autosomal recessive genetic disorder characterized by poikiloderma of the face, small stature, sparse scalp hair, juvenile cataract, radial aplasia, and predisposition to cancers. Due to the rarity of RTS, the situation of patients with RTS in Japan has not been elucidated. METHODS: In 2010 and 2020, following the results of a primary questionnaire survey, a secondary questionnaire survey on RTS was conducted nationwide to investigate the number of RTS cases and their associated skin lesions, bone lesions, other clinical features, and quality of life in Japan. RESULTS: In 2010 and 2020, 10 and eight patients with RTS were recruited, respectively. Skin lesions such as poikiloderma, erythema, pigmentation, and abnormal scalp hair were observed in almost all cases. Bone lesions were observed in four cases in the 2010 and 2020 surveys, respectively. Two cases had mutations in the RECQL4 gene in the 2020 survey. CONCLUSIONS: Two nationwide surveys have shown the actual situation of patients with RTS in Japan. Cutaneous and bone manifestations are important for the diagnosis of RTS. However, many patients have no RECQL4 mutations. The novel causative gene of RTS should be further elucidated.

Digenic mutations in ALDH2 and ADH5 impair formaldehyde clearance and cause a multisystem disorder, AMeD syndrome.

Rs671 in the aldehyde dehydrogenase 2 gene (ALDH2) is the cause of Asian alcohol flushing response after drinking. ALDH2 detoxifies endogenous aldehydes, which are the major source of DNA damage repaired by the Fanconi anemia pathway. Here, we show that the rs671 defective allele in combination with mutations in the alcohol dehydrogenase 5 gene, which encodes formaldehyde dehydrogenase (ADH5FDH ), causes a previously unidentified disorder, AMeD (aplastic anemia, mental retardation, and dwarfism) syndrome. Cellular studies revealed that a decrease in the formaldehyde tolerance underlies a loss of differentiation and proliferation capacity of hematopoietic stem cells. Moreover, Adh5-/-Aldh2 E506K/E506K double-deficient mice recapitulated key clinical features of AMeDS, showing short life span, dwarfism, and hematopoietic failure. Collectively, our results suggest that the combined deficiency of formaldehyde clearance mechanisms leads to the complex clinical features due to overload of formaldehyde-induced DNA damage, thereby saturation of DNA repair processes.

VGF nerve growth factor inducible is involved in retinal ganglion cells death induced by optic nerve crush.

VGF nerve growth factor inducible (VGF) is a polypeptide that is induced by neurotrophic factors and is involved in neurite growth and neuroprotection. The mRNA of the Vgf gene has been detected in the adult rat retina, however the roles played by VGF in the retina are still undetermined. Thus, the purpose of this study was to determine the effects of VGF on the retinal ganglion cells (RGCs) of mice in the optic nerve crush (ONC) model, rat-derived primary cultured RGCs and human induced pluripotent stem cells (iPSCs)-derived RGCs. The mRNA and protein of Vgf were upregulated after the ONC. Immunostaining showed that the VGF was located in glial cells including Müller glia and astrocytes but not in the retinal neurons and their axons. AQEE-30, a VGF peptide, suppressed the loss of RGCs induced by the ONC, and it increased survival rat-derived RGCs and promoted the outgrowth of neurites of rat and human iPSCs derived RGCs in vitro. These findings indicate that VGF plays important roles in neuronal degeneration and has protective effects against the ONC on RGCs. Thus, VGF should be considered as a treatment of RGCs degeneration.

A Docosahexaenoic Acid-Derived Pro-resolving Agent, Maresin 1, Protects Motor Neuron Cells Death.

Maresin 1 is a novel pro-resolving mediator derived from docosahexaenoic acid (DHA), with potent anti-inflammation effects against several animal models, including brain ischemia, sepsis, and lung fibrosis. However, its effect against motor neuron cell death is still not investigated. Therefore, we investigated the effects of maresin 1 on several stress-induced motor neuron cell death. Maresin 1 suppressed combinatorial stress which was evoked by superoxide dismutase 1 (SOD1)G93A and serum-free, -induced motor neuron cells death in a concentration-dependent manner, and had a stronger neuroprotective effective than DHA. Maresin 1 also had neuroprotective effects against transactivation response DNA-binding protein (TDP)-43A315T and serum-free stress, H2O2, and tunicamycin-induced cell death. Maresin 1 reduced the reactive oxygen species (ROS) production caused by SOD1G93A or TDP-43A315T. Moreover, maresin 1 suppressed the NF-κB activation induced by SOD1G93A and serum-free stress. These data indicate that maresin 1 has motor neuron protective effects against several stresses by reduction of ROS production or attenuation of the NF-κB activation. Maresin 1 also had neuroprotective effects against H2O2, and tunicamycin-induced cell death in a concentration-dependent manner. Finally, maresin 1 ameliorated the motor function deficits of spinal muscular atrophy model in which endoplasmic reticulum stress was upregulated. Thus, maresin 1 may be beneficial to protect against motor neuron diseases.

Loss of DNA Damage Response in Neuroblastoma and Utility of a PARP Inhibitor.

Background: Neuroblastoma (NB) is the most common solid tumor found in children, and deletions within the 11q region are observed in 11% to 48% of these tumors. Notably, such tumors are associated with poor prognosis; however, little is known regarding the molecular targets located in 11q. Methods: Genomic alterations of ATM , DNA damage response (DDR)-associated genes located in 11q ( MRE11A, H2AFX , and CHEK1 ), and BRCA1, BARD1, CHEK2, MDM2 , and TP53 were investigated in 45 NB-derived cell lines and 237 fresh tumor samples. PARP (poly [ADP-ribose] polymerase) inhibitor sensitivity of NB was investigated in in vitro and invivo xenograft models. All statistical tests were two-sided. Results: Among 237 fresh tumor samples, ATM, MRE11A, H2AFX , and/or CHEK1 loss or imbalance in 11q was detected in 20.7% of NBs, 89.8% of which were stage III or IV. An additional 7.2% contained ATM rare single nucleotide variants (SNVs). Rare SNVs in DDR-associated genes other than ATM were detected in 26.4% and were mutually exclusive. Overall, samples with SNVs and/or copy number alterations in these genes accounted for 48.4%. ATM-defective cells are known to exhibit dysfunctions in homologous recombination repair, suggesting a potential for synthetic lethality by PARP inhibition. Indeed, 83.3% NB-derived cell lines exhibited sensitivity to PARP inhibition. In addition, NB growth was markedly attenuated in the xenograft group receiving PARP inhibitors (sham-treated vs olaprib-treated group; mean [SD] tumor volume of sham-treated vs olaprib-treated groups = 7377 [1451] m 3 vs 298 [312] m 3 , P = .001, n = 4). Conclusions: Genomic alterations of DDR-associated genes including ATM, which regulates homologous recombination repair, were observed in almost half of NBs, suggesting that synthetic lethality could be induced by treatment with a PARP inhibitor. Indeed, DDR-defective NB cell lines were sensitive to PARP inhibitors. Thus, PARP inhibitors represent candidate NB therapeutics.

T-cell epitope-containing hypoallergenic ß-lactoglobulin for oral immunotherapy in milk allergy.

BACKGROUND: Optimally hydrolyzed ß-Lactoglobulin (ßLg) is a promising milk oral immunotherapy (OIT) candidate with respect to showing reduced B-cell reactivity but retaining the T-cell epitope. To demonstrate that an edible hypoallergenic ßLg hydrolysate containing the T-cell epitope is suitable for OIT. We tested how chymotrypsin affected the retention of the T-cell epitope of ßLg when preparing ßLg hydrolysates using food-grade trypsin. METHODS: We investigated the effect of chymotrypsin activity on the formation of the T-cell epitope-containing peptide of ßLg (ßLg102-124 ) and prepared an edible ßLg hydrolysate containing ßLg102-124 using screened food-grade trypsins. B-cell reactivity was determined using immunoassays in which ELISA was performed with anti-ßLg rabbit IgG and Western blotting was performed with a milk-specific IgE antiserum. RESULTS: In ßLg hydrolysis performed by varying the activity of trypsin and chymotrypsin, chymotrypsin activity inhibited the formation of ßLg102-124 with an increase in hydrolysis time in a dose-dependent manner. ßLg102-124 was generated by two of five food-grade trypsins used at a ratio of 1:50 (w/w, enzyme/substrate) for 20 h at 40°C. The edible ßLg hydrolysate retained ßLg102-124 and showed a reduction in molecular weight distribution and antigenicity against IgG and IgE. CONCLUSIONS: Chymotrypsin activity inhibited the formation of ßLg102-124 in the trypsin hydrolysate of ßLg. This ßLg trypsin hydrolysate is a novel candidate for peptide-based OIT in cow's milk allergy for safely inducing desensitization.

Indocyanine green lymphography and lymphaticovenous anastomosis for generalized lymphatic dysplasia with pleural effusion and ascites in neonates.

BACKGROUND: The fatality rate of generalized lymphatic dysplasia (GLD) with chylous pleural effusion and ascites is particularly high when it persists over a prolonged period. The purpose of this report was to determine the utility of indocyanine green (ICG) lymphography and lymphaticovenous anastomosis (LVA) in GLD with chylous pleural effusion and ascites in neonates. METHODS: We tested the lymphatic function in the 4 extremities for 8 GLD neonate patients using ICG lymphography, and on the basis of the results, we performed LVA for 5 of them. LVA was performed at the extremities under general anesthesia using incisions <1 cm in length. The outcome of LVA was evaluated with the amount of ascites discharged from thoracostomy tube or abdominal tube, except for 1 patient who had no drainage tube. RESULTS: In all cases, ICG lymphography showed varying degrees of dermal backflow in the limbs with lymphostasis. After LVA surgery, effusion stopped in 2 cases and decreased in 1 case. In the cases where effusion stopped, backflow as observed with ICG lymphography was minimal, and in the case where effusion decreased but did not stop, backflow was moderate. CONCLUSIONS: The application of ICG and LVA could possibly be used to diagnose and treat lymphatic pleural effusion or ascites.

Peripheral blood stem cell transplantation in a significant body weight difference between a smaller donor and a larger recipient: a case report.

Allogeneic peripheral blood stem cell transplantation (PBSCT) is becoming a common transplantation procedure in children. However, few benefits have been reported, in particular in regard to the choice of small children as donors for larger recipients. We report a case of relapsed acute myeloid leukemia (body weight 52 kg and blood type O) who underwent allogeneic PBSCT from his smaller human leukocyte antigen-matched brother (body weight 29.9 kg and blood type A).

Paclitaxel-based chemotherapy for aggressive kaposiform hemangioendothelioma of the temporomastoid region: Case report and review of the literature.

BACKGROUND: Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor of infancy and childhood. This tumor results in poor prognosis, and therefore, development of a more effective treatment is needed. METHODS AND RESULTS: We describe an 11-year-old boy presenting with left facial palsy caused by aggressive KHE of the left temporomastoid region. He was treated with paclitaxel-based chemotherapy, because of the difficulty with complete surgical resection for anatomic factor, multiple lung metastases on diagnosis, and no response to conventional treatments. This treatment reduced the volume of primary tumor and lung metastatic lesions, but the efficacy was transitory. CONCLUSIONS: Paclitaxel-based chemotherapy for aggressive KHE may be effective, therefore the multimodality therapy including paclitaxel of aggressive KHE, particularly in the head and neck, needs to be investigated in further studies.

In vitro analysis of the functional effects of an NLRP3 G809S variant with the co-existence of MEFV haplotype variants in atypical autoinflammatory syndrome.

PURPOSE: Hereditary periodic fever syndromes have been considered monogenic diseases. However, some recent reports have described patients with co-existence of recurrent fever responsible genes. This study assessed whether a rare variant, found in Japanese children showing atypical autoinflammatory syndrome, located in the leucine-rich repeat domain of Nod-like receptor family, pyrin domain containing 3 (NLRP3) with co-existence of Mediterranean fever (MEFV) haplotype variants may contribute to a proinflammatory phenotype using a systematic approach. METHODS: Cytokine production in serum or from peripheral blood monocytes was measured by ELISA. DNA sequence analysis of genes including NLRP3, MEFV, mevalonate kinase (MVK), and tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A) were performed on patient samples. In vitro functional assays determined the effects of the NLRP3 variants and pyrin using NF-κB activation and speck formation assays. RESULTS: A heterozygous genetic variant of NLRP3, G809S, was found in samples from both patients. Additionally the previously reported heterozygous MEFV variants (P369S-R408Q or E148Q-P369S-R408Q) were also detected in both patients. Serum IL-1ra and sTNFR1 levels increased in the attack phase of the disease in both patients. The production levels of IL-1ß from monocytes isolated from both cases were elevated following LPS and IFN-γ stimulation. The NLRP3 G809S variant demonstrated no increase of NF-κB activity following monosodium urate stimulation, whereas it significantly increased speck formation by interacting with apoptosis-associated speck-like protein with caspase recruitment domain. CONCLUSIONS: The phenotype of atypical autoinflammatory disease in patients could be modified by a synergistic effect with two other variants of autoinflammatory-associated genes.

Autosomal-dominant chronic mucocutaneous candidiasis with STAT1-mutation can be complicated with chronic active hepatitis and hypothyroidism.

PURPOSE: To describe a case of autosomal-dominant (AD)-chronic mucocutaneous candidiasis (CMC) with a signal transducer and activator of transcription (STAT) 1 gene mutation, and some of the important complications of this disease such as chronic hepatitis. METHODS: We present a 23-year-old woman with CMC, chronic active hepatitis, and hypothyroidism. Her father also had CMC. We performed several immunological analyses of blood and liver samples, and searched for gene mutations for CMC in the patient and her father. RESULTS: We identified the heterozygous substitution c.821 G > A (p.Arg274Gln) in the STAT1 gene of both the patient and her father. The level of ß-glucan induced interferon (IFN)-γ in her blood cells was significantly low. Immunoblot analysis detected serum anti-interleukin (IL)-17 F autoantibody. She was found to have increased (low-titer) antibodies related to her hypothyroidism and hepatitis. Her serum IL-18 levels fluctuated with her AST and ALT levels. Liver biopsy revealed CD68-positive cell infiltration and IL-18 expression in the sinusoidal regions. These results suggest that the chronic active hepatitis in this patient may be exacerbated by the excessive IL-18 accumulation caused by recurrent mucocutaneous fungal infection, and decreased IFN-γ production. CONCLUSIONS: AD-CMC is known to be caused by a gain-of-function mutation of the STAT1 gene. Chronic active hepatitis is a rare complication of AD-CMC, with currently unknown pathogenesis. It seems that the clinical phenotype in this patient is modified by autoimmune mechanisms and cytokine dysregulation. AD-CMC can be complicated by various immune disorders including autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy.

Characterization of NLRP3 variants in Japanese cryopyrin-associated periodic syndrome patients.

The etiology of cryopyrin-associated periodic syndrome (CAPS) is caused by germline gene mutations in NOD-like receptor family, pryin domain containing 3 (NLRP3)/cold-induced autoinflammatory syndrome 1 (CIAS1). CAPS includes diseases with various severities. The aim of this study was to characterize patients according to the disease severity of CAPS. Five Japanese patients with four kinds of gene variations in NLRP3 were found and diagnosed as CAPS or juvenile idiopathic arthritis. Two mutations in NLRP3, Y563N and E688K, found in CAPS patients exhibit significant positive activities in the nuclear factor-κB reporter gene assay. Increased serum interleukin (IL)-18 levels were only observed in severe cases of CAPS. In mild cases of CAPS, the serum IL-18 levels were not increased, although lipopolysaccharide- or hypothermia-enhanced IL-1ß and IL-18 production levels by their peripheral blood mononuclear cells were detectable. This series of case reports suggests that a combination of in vitro assays could be a useful tool for the diagnosis and characterization of the disease severity of CAPS.

Adipokine ganglioside GM2 activator protein stimulates insulin secretion.

Recently, we identified ganglioside GM2 activator protein (GM2AP) as a novel adipokine, and revealed that treatment of cultured cells with GM2AP impairs insulin signal transduction. The aim of this study was to examine the impact of GM2AP on glucose metabolism in vivo. Injection of recombinant GM2AP in mice significantly lowered blood glucose levels in glucose tolerance tests. Administration of GM2AP to mice for 10 days increased serum insulin levels, whereas the contents of glucose, leptin and FFA were significantly decreased. Stimulation of calcium influx and insulin secretion by GM2AP was observed in hamster insulinoma HIT-T15 cells. Blockage of GM2AP function by specific antibodies inhibited GM2AP-induced insulin secretion. These results provide novel insights into the physiological functions of GM2AP in obesity.

Augmented cell death with Bloom syndrome helicase deficiency.

Bloom syndrome (BS) is a rare autosomal genetic disorder characterized by lupus-like erythematous telangi-ectasias of the face, sun sensitivity, infertility, stunted growth, upper respiratory infection, and gastrointestinal infections commonly associated with decreased immuno-globulin levels. The syndrome is associated with immuno-deficiency of a generalized type, ranging from mild and essentially asympto-matic to severe. Chromosomal abnormalities are hallmarks of the disorder, and high frequencies of sister chromatid exchanges and quadriradial configurations in lymphocytes and fibroblasts are diagnostic features. BS is caused by mutations in BLM, a member of the RecQ helicase family. We determined whether BLM deficiency has any effects on cell growth and death in BLM-deficient cells and mice. BLM-deficient EB-virus-transformed cell lines from BS patients and embryonic fibroblasts from BLM-/- mice showed slower growth than wild-type cells. BLM-deficient cells showed abnormal p53 protein expression after irradiation. In BLM-/- mice, small body size, reduced number of fetal liver cells and increased cell death were observed. BLM deficiency causes the up-regulation of p53, double-strand break and apoptosis, which are likely observed in irradiated control cells. Slow cell growth and increased cell death may be one of the causes of the small body size associated with BS patients.

A novel adipokine GM2AP impairs insulin signaling.

In an attempt to discover novel adipokines, we performed proteomics analyses using culture medium from differentiated 3T3-L1 adipocytes, and first identified GM2AP. The levels of GM2AP mRNA and protein were augmented by adipogenesis in cultured adipocytes and expression in adipose tissue and serum of obese mice or human subjects was found to be significantly higher than in lean counterparts. Exposure of 3T3-L1 adipocytes to GM2AP protein accelerated dissociation of insulin receptor-beta (IRß) from caveolin-1, and interrupted insulin signal transduction. Abrogation of GM2AP function by specific antibodies augmented glucose uptake. Furthermore, treatment of rat pheochromocytoma PC12 NS1 cells with GM2AP impaired NGF signal transduction. Taken together, these results provide novel insights into the physiological functions of GM2AP in obesity.

Reversible cerebrospinal fluid edema and porencephalic cyst, a rare complication of ventricular catheter.

Cerebrospinal fluid (CSF) edema and porencephaly are rare postoperative complications of a ventricular shunt that result from obstruction of the distal catheter, especially in children with taut ventricles. We report a 10-year-old male with cerebellar germinoma complicated by obstructive hydrocephalus. Ventriculopuncture was performed and an Ommaya reservoir was implanted at the right frontal horn. A distal catheter was initially attached to the reservoir for drainage of hydrocephalus but was later removed. After surgery, multi-agent chemotherapy and radiation therapy, a brain MRI showed CSF edema and porencephaly in the right frontal white matter. These lesions were reduced by prompt removal of the ventricular catheter. It is important to recognize such complications and to remove the catheter as soon as possible, because the brain tissue affected by massive edema may develop irreversible changes. Advanced MRI techniques, including fluid-attenuated inversion recovery (FLAIR) and diffusion-weighted imaging may be helpful for assessing this pathological condition and its prognosis.

Metabolism of 2,6-dichloro-4-(3,3-dichloroallyloxy)phenyl 3-[5-(trifluoromethyl)-2-pyridyloxy]propyl ether (pyridalyl) in rats after repeated oral administration and a simple physiologically based pharmacokinetic modeling in brown and white adipose tissues.

Male and female Sprague-Dawley rats received repeated oral administration of 14C-2,6-dichloro-4-(3,3-dichloroallyloxy)phenyl 3- [5-(trifluoromethyl)-2-pyridyloxy]propyl ether (14C-pyridalyl) at 5 mg/kg/day for 14 consecutive days, and 14C excretion, 14C concentration in tissues, and the metabolic fate were determined. Most 14C was excreted into feces. The 14C concentrations in the blood and tissues attained steady-state levels at days 6 to 10, whereas those in white adipose tissues increased until day 14. Tissue 14C concentrations were highest in brown and white adipose tissue (38.37-57.50 ppm) but were 5.60 ppm or less in all the other tissues. Total 14C residues in blood and tissues on the 27th day after the first administration accounted for 2.6 to 3.2% of the total dose. A major fecal metabolite resulted from O-dealkylation. Analysis of metabolites in tissues revealed that the majority of 14C in perirenal adipose tissue and lungs was pyridalyl, accounting for greater than 90 and 60%, respectively, of the total, whereas a major metabolite in whole blood, kidneys, and liver was a dehalogenated metabolite. The experimental data were simulated with simple physiologically based pharmacokinetics using four-compartment models with assumption of lymphatic absorption and membrane permeability in adipose tissues. The different kinetics in brown and white adipose tissues was reasonably predicted in this model, with large distribution volume in adipose tissues and high hepatic clearance in liver. Sex-related difference of pyridalyl concentration in liver was considered to be a result of different unbound fraction times the hepatic intrinsic clearance (f x CL(int)) of 1.8 and 12 l/h for male and female, respectively.