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Phelan-McDermid syndrome (PMS) is a 22q13.3 deletion syndrome. Most PMS patients show global developmental delay and some of them suffer from developmental regression. The deleted region contains ARSA, which is responsible for metachromatic leukodystrophy (MLD). Here we report an extremely rare case of PMS characterized by unusual, rapidly progressive developmental regression due to additional pathogenic mutation in ARSA. Considering the 1 in 100 chance of an MLD carrier, co-occurrence of PMS and MLD in a patient is possible if either parent carries a heterozygous ARSA mutation. Therefore, MLD should be ruled out in PMS patients with severe neurological phenotype.

Background: The expression of major histocompatibility complex class I (MHC I) has previously been reported to be negatively associated with estrogen receptor (ER) expression. Furthermore, the expression of MHC I, the level of tumor-infiltrating lymphocytes (TILs), and the expression of interferon (IFN) mediator MxA are positively associated with one another in human breast cancers. This study aimed to investigate the mechanisms of association of MHC I with estrogen and IFN signaling. Methods: The human leukocyte antigen (HLA)-ABC protein expression was analyzed in breast cancer cell lines. The expressions of HLA-A and MxA mRNAs were analyzed in MCF-7 cells in Gene Expression Omnibus (GEO) data. ER and HLA-ABC expressions and TIL levels in tumor tissue were also analyzed in ER+/ human epidermal growth factor receptor 2 (HER2)- breast cancer patients who randomly received either neoadjuvant chemotherapy or estrogen modulator treatment following surgical resection. Results: HLA-ABC protein expression was decreased after β-estradiol treatment or hESR-GFP transfection and increased after fulvestrant or IFN-γ treatment in breast cancer cell lines. In GEO data, HLA-A and MxA expression was increased after ESR1 shRNA transfection. When comparing the two patient groups, ER Allred score was significantly lower and the HLA-ABC expression and TIL levels were significantly higher in the estrogen modulator treated group than the chemotherapy treated group. Conclusion: MHC I expression and TIL levels might be affected by ER pathway modulation and IFN treatment. Further studies elucidating the mechanism of MHC I regulation could suggest a way to boost TIL influx in cancer in a clinical setting.

BackgroundAmbroxol (ABX) has been suggested as an augmentative pharmacological agent for neuronopathic Gaucher disease (nGD). This study assessed the long-term safety and efficacy of combined therapy with high-dose ABX and enzyme replacement therapy (ERT) in nGD.MethodsABX+ERT therapy was administered for 4.5 years in four patients with nGD. ABX was initiated at a dose of 1.5 mg/kg/day, and the dose was escalated up to 27 mg/kg/day. The target plasma level was 10 µmol/L or less. The changes in glucocerebrosidase activity, biochemical, safety and neurocognitive findings were assessed.ResultsEnhanced residual GCcase activity was observed in all patients, as evidenced in both in vitro and in vivo studies. During the first 2 years of study with ABX (up to 21 mg/kg/day), mean seizure frequencies and neurocognitive function worsened. After ABX dosage was increased up to 27 mg/kg/day of ABX, its trough plasma concentration was 3.2–8.8 µmol/L. Drug-to-drug interaction, especially with antiepileptic drug significantly affected the pharmacokinetic parameters of ABX. Importantly, at 27 mg/kg/day of ABX, the seizure frequencies markedly decreased from the baseline, and the neurocognitive function was improved. In addition, Lyso-Gb1, a biomarker for the severity and progression of GD, was normalised in all patients. High-dose ABX was well-tolerated with no severe adverse events.ConclusionsLong-term treatment with high-dose ABX+ERT was safe and might help to arrest the progression of the neurological manifestations in GD.

Lesch-Nyhan syndrome (LNS) is an X-linked recessive disorder caused by mutations in the HPRT1 gene. The clinical features and mutation spectrum of 26 Korean LNS patients from 23 unrelated families were retrospectively reviewed. The HPRT1 gene was analyzed by direct sequencing of genomic DNA. The median age at diagnosis was 2.3 years (range, 4 months-22.6 years) and the initial presenting features included developmental delay, orange colored urine, and self-injurious behaviors. Most patients were wheelchair-bound and suffered from urinary complications and neurologic problems such as self-mutilation and developmental delay. Twenty different mutations in HPRT1 were identified among 23 independent pedigrees, including six novel mutations. The most common mutation type was truncating mutations including nonsense and frameshift mutations (45%). Large deletions in the HPRT1 gene were identified in exon 1, exons 5-6, exons 1-9, and at chr X:134,459,540-134,467,241 (7702 bp) including the 5'-untranslated region, exon 1, and a portion of intron 1. In conclusion, this study describes the phenotypic spectrum of LNS and has identified 20 mutations from 23 Korean families, including six novel mutations in Korean patients with LNS.

(1) Background: Pancreatic cancer is a high devastating disease with the lowest survival rate among all common cancers due to difficulties in early diagnosis. The purpose of this study was to identify and characterize the distinct subset of blood cell population elevated in peripheral blood mononuclear cells (PBMC) of pancreatic cancer to evaluate the potential markers for diagnosis of pancreatic cancer, (2) Methods: We analyzed differential gene expression in PBMC from normal individuals and pancreatic cancer patients utilizing transcriptome analysis. Flow cytometry analysis was applied to identify the discrete subset of interleukin-7 receptor (IL-7R) expressing cells in these cells. The expression of IL-7R during tumorigenesis was determined in syngeneic mouse model of pancreatic cancer in vivo, (3) Results: PBMC from pancreatic cancer patients expressed elevated IL-7R mRNA compared to healthy control individuals. IL-7R expressing cells rapidly appeared from the early stages of the onset of tumor formation in syngeneic pancreatic cancer mouse model in vivo. The discrete subset of IL-7R positive cells mainly consist of naïve T, central memory T, and effector memory T cells, (4) Conclusions: Taken together, our present findings suggest that pancreatic cancer patients expressed higher level of IL-7R expression in PBMC that rapidly emerged from the onset of early pancreatic tumor formation in vivo than normal individuals. Thus, it can be used as a novel biological marker for early events of pancreatic cancer development.

Schaaf-Yang syndrome (SYS) is a recently identified disorder caused by a loss-of-function mutation in a maternally imprinted gene, MAGEL2, at 15q11.2q13. Due to its extreme rarity and wide range of clinical severity, clinical suspicion is difficult for a physician. In the current study, its frequency among the Korean pediatric patients with developmental delay (DD) or intellectual disability (ID) was assessed. As the first report of Korean patients with SYS, our study aims to increase the awareness of this condition among the physicians taking care of the pediatric patients with DD/ID and hypotonia. The patients diagnosed with SYS by whole-exome sequencing (WES) among the 460 Korean pediatric patients with DD/ID were included, and their clinical and molecular features were reviewed. Four patients (0.9%) were diagnosed with SYS. Profound DD (4 patients), multiple anomalies including joint contractures and facial dysmorphism (4 patients), generalized hypotonia (3 patients), and severe respiratory difficulty requiring mechanical ventilation (3 patients) were noted in most cases, similar to those in previous reports. Sleep apnea (2 patients), autistic features (2 patients), a high grade of gastroesophageal reflux (1 patient), and seizures (1 patient) were found as well. A total of 3 different truncating MAGEL2 mutations were identified. A previously-reported mutation, to be the most common one, c.1996dupC, was found in 2 patients. The other 2 mutations, c.2217delC and c.3449_3450delTT were novel mutations. As MAGEL2 is maternally imprinted, 2 patients had inherited the MAGEL2 mutation from their respective healthy fathers. SYS is an extremely rare cause of DD/ID. However, hypotonia, joint contractures, profound DD/ID and facial dysmorphism are the suggestive clinical features for SYS. As a maternally imprinted disorder, it should be reminded that SYS may be inherited in form of a mutation from a healthy father.

Background Gaucher disease (GD) is caused by a deficiency of β-glucocerebrosidase, encoded by GBA. Haplotype analyses previously demonstrated founder effects for particular GBA mutations in Ashkenazi Jewish and French-Canadian populations. This study aimed to investigate the clinical characteristics and mutation spectrum of GBA in Korean GD patients and to identify founder effect of GBA p.G85E in non-neuronopathic GD patients. Results The study cohort included 62 GD patients from 58 unrelated families. Among them, 18 patients from 17 families harbored the p.G85E mutation. Haplotype analysis was performed for 9 probands and their parents for whom DNA samples were available. In 58 unrelated probands, the GBA mutation p.L483P was the most common (30/116 alleles, 26%), followed by p.G85E (16%), p.F252I (13%), and p.R296Q (9%). The median age at diagnosis of the 18 patients harboring the p.G85E mutation was 3.8 (range 1.2–57) years. No patients developed neurological symptoms during follow-up periods of 2.2–20.3 (median 13.9) years. The size of the shared haplotype containing GBA p.G85E was 732 kbp, leading to an estimated age of 3075 years. Conclusion The GBA p.G85E mutation, which appears to be neuroprotective despite producing distinctive visceromegaly and skeletal symptoms, exhibited a potential founder effect in Korean GD patients.

Purpose In the presence of interferon, proteasome subunits are replaced by their inducible counterparts to form an immunoproteasome (IP) plays a key role in generation of antigenic peptides presented by MHC class I molecules, leading to elicitation of a T cell‒mediated immune response. Although the roles of IP in other cancers, and inflammatory diseases have been extensively studied, its significance in breast cancer is unclear. Materials and Methods We investigated the expression of LMP7, an IP subunit, and its relationship with immune system components in two breast cancer cohorts. Results In 668 consecutive breast cancer cohort, 40% of tumors showed high level of LMP7 expression, and tumors with high expression of LMP7 had more tumor-infiltrating lymphocytes (TILs) in each subtype of breast cancer. In another cohort of 681 triple-negative breast cancer patients cohort, the expression of LMP7 in tumor cells was significantly correlated with the amount of TILs and the expression of interferon-associated molecules (MxA [p < 0.001] and PKR [p < 0.001]), endoplasmic reticulum stress-associated molecules (PERK [p=0.012], p-eIF2a [p=0.001], and XBP1 [p < 0.001]), and damage-associated molecular patterns (HMGN1 [p < 0.001] and HMGB1 [p < 0.001]). Patients with higher LMP7 expression had better disease-free survival outcomes than those with no or low expression in the positive lymph node metastasis group (p=0.041). Conclusion Close association between the TIL levels and LMP7 expression in breast cancer indicates that better antigen presentation through greater LMP7 expression might be associated with more TILs.

Adoptive cell transfer (ACT) of ex vivo expanded tumor-infiltrating lymphocytes (TILs) has been successful in treating a considerable proportion of patients with metastatic melanoma. In addition, some patients with several other solid tumors were recently reported to have benefited clinically from such ACT. However, it remains unclear whether ACT using TILs is broadly applicable in breast cancer, the most common cancer in women. In this study, the utility of TILs as an ACT source in breast cancers was explored by deriving TILs from a large number of breast cancer samples and assessing their biological potentials. We successfully expanded TILs ex vivo under a standard TIL culture condition from over 100 breast cancer samples, including all breast cancer subtypes. We also found that the information about the percentage of TIL and presence of tertiary lymphoid structure in the tumor tissues could be useful for estimating the number of obtainable TILs after ex vivo culture. The ex vivo expanded TILs contained a considerable level of central memory phenotype T cells (about 20%), and a large proportion of TIL samples were reactive to autologous tumor cells in vitro. Furthermore, the in vitro tumor-reactive autologous TILs could also function in vivo in a xenograft mouse model implanted with the primary tumor tissue. Collectively, these results strongly indicate that ACT using ex vivo expanded autologous TILs is a feasible option in treating patients with breast cancer.

PURPOSE Congenital hypopituitarism is caused by mutations in pituitary transcription factors involved in the development of the hypothalamic-pituitary axis. Mutation frequencies of genes involved in congenital hypopituitarism are extremely low and vary substantially between ethnicities. This study was undertaken to compare the clinical, endocrinological, and radiological features of patients with an isolated growth hormone deficiency (IGHD) or combined pituitary hormone deficiency (CPHD). MATERIALS AND METHODS This study included 27 patients with sporadic IGHD and CPHD. A mutation analysis of the POU1F1, PROP1, LHX3, LHX4, and HESX1 genes was performed using genomic DNA from peripheral blood leukocytes. RESULTS IGHD and CPHD were observed in 4 and 23 patients, respectively. Mean age at diagnosis was 8.28±7.25 years for IGHD and 13.48±10.46 years for CPHD (p=0.37). Serum insulin-like growth factor-1 and peak growth hormone (GH) levels following GH stimulation tests were significantly lower in patients with CPHD than in those with IGHD (p

// Sun-Young Kong 2, 3, 4 , Kwang-Soo Kim 1 , Jiewan Kim 1 , Min Kyeong Kim 2 , Ki Hong Lee 2 , Je-Yong Lee 1 , Nuri Oh 1 , Ji-In Park 1 , Ji-Hoon Park 1 , Sun-Hee Heo 5 , Sung Han Shim 1 , Dong Ryul Lee 1 , Keun Pil Kim 6 , Kyung-Soon Park 1 1 Department of Biomedical Science, College of Life Science, CHA University, Seoul, Korea 2 Department of System Cancer Science, Graduate School of Cancer Science and Policy, National Cancer Center, Seoul, Korea 3 Translational Epidemiology Branch, Research Institute, National Cancer Center, Seoul, Korea 4 Department of Laboratory Medicine, Center for Diagnostic Oncology, Hospital, National Cancer Center, Seoul, Korea 5 Biomedical Research Center, Asan Institute for Science and Department of Pathology, Asan Medical Center, University of Ulsan, College of Medicine, Ulsan, Korea 6 Department of Life Science, Chung-Ang University, Seoul, Korea Correspondence to: Kyung-Soon Park, email: kspark@cha.ac.kr Keywords: ELK3, GATA3, metastasis, migration, invasion Received: February 02, 2016 Accepted: August 13, 2016 Published: August 19, 2016 ABSTRACT Triple-negative breast cancer is a highly aggressive tumor subtype that lacks effective therapeutic targets. Here, we show that ELK3 is overexpressed in a subset of breast cancers, in particular basal-like and normal-like/claudin-low cell lines. Suppression of ELK3 in MDA-MB-231 cells led to transdifferentiation from an invasive mesenchymal phenotype to a non-invasive epithelial phenotype both in vitro and in vivo . Suppression of ELK3 resulted in extensive changes in genome expression profiles. Among these, GATA3, a master suppressor of metastasis, was epigenetically activated. Also, suppression of GATA3 led to the restoration of migration and invasion. These results suggest that the ELK3-GATA3 axis is a major pathway that promotes metastasis of MDA-MB-231 cells.

Objectives: We tried to describe cellular immune response (tertiary lymphoid structures (TLSs), lymphoid aggregates, tumor infiltrating lymphocytes (TILs)) in neoplastic microenvironment of ductal carcinoma in situ (DCIS) with or without associated microinvasion. Methods: The histopathologic parameters of 177 DCIS and 27 DCIS with microinvasion were evaluated. We determined number of ducts involved by DCIS, and calculated percentage of these ducts surrounded by TLSs. TILs were quantitated in 27 microinvasive cases. Results: Tumors having higher percentage of DCIS ducts associated with TLSs had higher incidence of microinvasion ( P

// Hee Jin Lee 1,* , In Hye Song 1,* , In Ah Park 1 , Sun-Hee Heo 1 , Young-Ae Kim 1 , Jin-Hee Ahn 2 and Gyungyub Gong 1 1 Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea 2 Department of Oncology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea * These authors have contributed equally to this work Correspondence to: Gyungyub Gong, email: // Keywords : breast carcinoma; tumor-infiltrating lymphocytes; major histocompatibility complex I; human leukocyte antigen; Pathology Section Received : December 14, 2015 Accepted : April 03, 2016 Published : April 18, 2016 Abstract Tumor-infiltrating lymphocytes (TILs) in triple-negative breast cancer (TNBC) have a strong prognostic and predictive significance. However, the mechanism of TIL influx in TNBC is unclear. Expression of major histocompatibility complex class I (MHC I) on the tumor cell is essential for the effective killing of tumor by cytotoxic TILs. In our current study, human leukocyte antigen (HLA) expression was inversely correlated with estrogen receptor (ER) expression in normal and cancerous breast tissue and positively correlated with TILs in breast cancer. The ER score was inversely correlated with TILs in breast cancer. HLA-A and CD8B gene expression was negatively correlated with ESR1 and positively correlated with interferon-associated gene expression in The Cancer Genome Atlas (TCGA) data. Negative correlation between ESR1 and HLA and positive correlation between interferon-associated and HLA gene expression were also confirmed in Cancer Cell Line Encyclopedia (CCLE) data. Taken together, our data suggest that a lower expression of HLA in luminal-type tumors might be associated with low level of TILs in those tumors. Further investigation of the mechanism of higher HLA expression and TIL influx in TNBC may help to boost the host immune response.

BACKGROUND Most triple-negative breast cancers (TNBCs) have a high histologic grade, are associated with high endoplasmic stress, and possess a high frequency of TP53 mutations. TP53 missense mutations lead to the production of mutant p53 protein and usually show high levels of p53 protein expression. Tumor-infiltrating lymphocytes (TILs) accumulate as part of the anti-tumor immune response and have a strong prognostic and predictive significance in TNBC. We aimed to elucidate the association between p53 expression and the amount of TILs in TNBC. METHODS In 678 TNBC patients, we evaluated TIL levels and expression of endoplasmic stress molecules. Immunohistochemical examination of p53 protein expression was categorized into three groups: no, low, and high expression. RESULTS No, low, and high p53 expression was identified in 44.1% (n = 299), 20.1% (n = 136), and 35.8% (n = 243) of patients, respectively. Patients with high p53 expression showed high histologic grade (p < .001), high TIL levels (p = .009), and high expression of endoplasmic reticulum stress-associated molecules (p-eIF2a, p = .013; XBP1, p = .007), compared to patients with low p53 expression. There was no significant difference in disease-free (p = .406) or overall survival rates (p = .444) among the three p53 expression groups. CONCLUSIONS High p53 expression is associated with increased expression of endoplasmic reticulum stress molecules and TIL influx.

The level of tumor-infiltrating lymphocytes and presence of tertiary lymphoid structures are significant prognostic and predictive factors in primary breast cancer. However, the understanding about differences in tumor-infiltrating lymphocytes and tertiary lymphoid structures at various metastatic sites or between primary breast tumors and metastatic sites is limited. A total of 335 cases of metastatic breast cancer from four metastatic sites (lung, liver, brain, and ovary) were included. We analyzed the percentages of tumor-infiltrating lymphocytes and presence of tertiary lymphoid structures in the primary and metastatic sites. The mean level of tumor-infiltrating lymphocytes in the lung metastases was higher than in the liver, brain, ovary, and matched primary tumors, while metastatic tumors of the liver and brain showed lower levels of tumor-infiltrating lymphocytes than primary tumors. Tertiary lymphoid structures were only found in the lung and liver, and in cases of brain metastases the change of tertiary lymphoid structures from present to absent significantly affected the level of tumor-infiltrating lymphocytes in metastases compared with that in matched primary tumors. Patients with a lower histological grade, hormone receptor positivity in primary tumors and metastases, a lower level of tumor-infiltrating lymphocytes and absence of tertiary lymphoid structures in primary tumors, a higher level of tumor-infiltrating lymphocytes and presence of tertiary lymphoid structures in metastases, and lung metastases showed significantly better overall survival. Our results showed that metastatic breast tumors in the lung had more tumor-infiltrating lymphocytes than did tumors at other sites and matched primary tumors. In addition, the presence of tertiary lymphoid structures in metastatic sites is a critical factor for the level of tumor-infiltrating lymphocytes.

Background Fatty acid oxidation disorders (FAODs) include more than 15 distinct disorders with variable clinical manifestations. After the introduction of newborn screening using tandem mass spectrometry, early identification of FAODs became feasible. This study describes the clinical, biochemical and molecular characteristics of FAODs patients detected by newborn screening (NBS) compared with those of 9 patients with symptomatic presentations. Methods Clinical and genetic features of FAODs patients diagnosed by NBS and by symptomatic presentations were reviewed. Results Fourteen patients were diagnosed with FAODs by NBS at the age of 54.8 ± 4.8 days: 5 with very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency, 5 with medium chain acyl-CoA dehydrogenase (MCAD) deficiency, 1 with primary carnitine deficiency, 1 with carnitine palmitoyltransferase 1A (CPT1A) deficiency, 1 with long-chain 3-hydroxyacyl-CoA dehydrogenase or mitochondrial trifunctional protein (LCAHD/MTP) deficiency, and 1 with short chain acyl-CoA dehydrogenase (SCAD) deficiency. Three patients with VLCAD or LCHAD/MTP deficiency developed recurrent rhabdomyolysis or cardiomyopathy, and one patient died of cardiomyopathy. The other 10 patients remained neurodevelopmentally normal and asymptomatic during the follow-up. In 8 patients with symptomatic presentation, FAODs manifested as LCHAD/MTP deficiencies by recurrent rhabdomyolysis or cadiomyopathy (6 patients), and VLCAD deficiency by cardiomyopathy (1 patient), and CPT1A deficiency by hepatic failure (1 patient). Two patients with LCHAD/MTP deficiencies died due to severe cardiomyopathy in the neonatal period, and developmental disability was noted in CPT1A deficiency (1 patient). Conclusions NBS helped to identify the broad spectrum of FAODs and introduce early intervention to improve the clinical outcome of each patient. However, severe clinical manifestations developed in some patients, indicating that careful, life-long observation is warranted in all FAODs patients.

Background: Ambroxol (ABX) has been suggested as an augmentative pharmacological agent for neuronopathic Gaucher disease (nGD). This study assessed the long-term safety and efficacy of combined therapy with high-dose ABX and enzyme replacement therapy (ERT) in nGD. Methods: Four patients with nGD were enrolled. ABX ERT therapy was administered for 4·5 years. Ambroxol was initiated at a dose of 1·5 mg/kg/day, and the dose was escalated up to 27 mg/kg/day. The changes in glucocerebrosidase (GBA) activity, biochemical, safety, and neurocognitive findings were assessed. Findings: Enhanced residual GBA activity was observed in all patients, as evidenced in both in vitro and in vivo studies. Mean seizure frequencies markedly decreased from the baseline, and the neurocognitive function was maintained. Lyso-Gb1, a biomarker for the severity and progression of GD, gradually decreased in all patients. High-dose ABX was well-tolerated with no severe adverse events. Interpretation: Long-term treatment with high-dose ABX ERT was safe and shows promise for arresting the progression of the neurological manifestations in GD. Clinical Trial Number: The study protocol was registered at the Clinical Research Information Service, Korea Centers for Disease Control and Prevention, Ministry of Health and Welfare (Republic of Korea) (no. KCT0003218). Funding Statement: This research was supported in part by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (NRF- 2015K1A4A3046807, NRF-2016M3A9B4915706 and NRF-2018M3A9H1078335) and by ISU ABXIS, Gyeonggi-do, Korea. Declaration of Interests: "None declared." Ethics Approval Statement: The study protocol was approved by the Institutional Review Board and the Ministry of Food and Drug Safety, Korea (no. 30449).

Nonimmune hydrops fetalis is the most severe clinical manifestation of lysosomal storage diseases (LSDs). Around 14 different LSDs have been accounted for as 1–15% of the cause of nonimmune hydrops fetalis. We report a Korean infant affected by an extremely rare but severe form of sialic acid storage disease. The patient presented with nonimmune hydrops fetalis, dysmorphic facial features, hepatosplenomegaly, and dysostosis multiplex and died at 39 days of age due to persistent pulmonary hypertension. LSD was suspected based on the presence of diffuse vacuolation of syncytiotrophoblast, villous stromal cells, and intermediate trophoblast in placental biopsy. Increased excretion of urinary free sialic acid was detected by liquid chromatography-tandem mass spectrometry. The patient was compound heterozygous of the c.908G>A (p.Trp303Ter) and the splicing mutation c.1259+5G>T (IVS9+5 G>T) in the SLC17A5 gene.

Supplemental Digital Content is available in the text, Fabry disease is a rare X-linked lysosomal storage disorder caused by an α-galactosidase A deficiency. The progressive accumulation of globotriaosylceramide (GL-3) results in life-threatening complications, including renal, cardiac, and cerebrovascular diseases. This study investigated the phenotypic and molecular spectra of GLA mutations in Korean patients with Fabry disease using a nationwide survey. This study included 94 patients from 46 independent pedigrees: 38 adult males, 46 symptomatic females, and 10 pediatric males. Each diagnosis was based on an enzyme assay and GLA gene mutation analysis. The mean age at presentation was 24 years (range, 5–65 years); however, the diagnoses were delayed by 21 ± 19 years after the onset of symptoms. Those patients with late-onset Fabry disease were diagnosed by family screening or milder symptoms at a later age. Forty different mutations were identified: 20 missense (50%), 10 nonsense (25%), 8 frameshift (20%), and 2 splice site (5%) mutations. Five of them were novel. IVS4+919G>A (c.936+919 G>A) was not detected among the 6505 alleles via newborn screening using dried blood spots. Enzyme replacement therapy (ERT) was performed in all the males and pediatric patients, whereas 75% of the symptomatic females underwent ERT for 4.2 ± 3.6 years. This study described the demographic data, wide clinical spectrum of phenotypes, and GLA mutation spectrum of Fabry disease in Korea. Most of the patients had classical Fabry disease, with a 4 times higher incidence than that of late-onset Fabry disease, indicating an underdiagnosis of mild, late-onset Fabry disease.

Background Fabry disease is characterised by the progressive accumulation of globotriaosylceramide (Gb3) and related glycosphingolipids in vascular endothelial cells. Enzyme replacement therapy (ERT) clears this accumulation. We analysed plasma proteome profiles before and after ERT to characterise its molecular pathology. Methods Two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation-time of flight tandem mass spectrometry (MALDI-TOF MS) and tandem mass spectrometry (MS/MS) were done using plasma samples before and after ERT in eight patients with classical Fabry disease Results After short-term ERT (4–12 months), the levels of 15 plasma proteins involved in inflammation, oxidative and ischaemic injury, or complement activation were reduced significantly. Among them, β-actin (ACTB), inactivated complement C3b (iC3b), and C4B were elevated significantly in pre-ERT Fabry disease plasma compared with control plasma. After longer-term ERT (46–96 months), iC3b levels gradually decreased, whereas the levels of other proteins varied. The gradual reduction of iC3b was comparable to that of Gb3 levels. In addition, iC3b increased significantly in pre-ERT Fabry disease mouse plasma, and C3 deposits were notable in renal tissues of pre-enzyme replacement therapy patients. Conclusion These results indicated that C3-mediated complement activation might be altered in Fabry disease and ERT might promote its stabilisation.

Theranostic agents present a promising clinical approach for cancer detection and treatment. We herein introduce a microbubble and liposome complex (MB-Lipo) developed for ultrasound (US) imaging and activation. The MB-Lipo particles have a hybrid structure consisting of a MB complexed with multiple Lipos. The MB components are used to generate high echo signals in US imaging, while the Lipos serve as a versatile carrier of therapeutic materials. MB-Lipo allows high contrast US imaging of tumor sites. More importantly, the application of high acoustic pressure bursts MBs, which releases therapeutic Lipos and further enhances their intracellular delivery through sonoporation effect. Both imaging and drug release could thus be achieved by a single US modality, enabling in situ treatment guided by real-time imaging. The MB-Lipo system was applied to specifically deliver anti-cancer drug and genes to tumor cells, which showed enhanced therapeutic effect. We also demonstrate the clinical potential of MB-Lipo by imaging and treating tumor in vivo.

Type 1 citrullinemia (CTLN1) often presents as a hyperammonemic encephalopathy in the neonatal period, but it can also develop in the late-infantile period and in adults. In addition, some patients can be identified in the presymptomatic period by neonatal or family member screening. In this study, twenty Korean patients with CTLN1 (19 families) were examined; fourteen patients with neonatal-onset, three with late-onset, and three that were identified presymptomatically. The 13 patients with hyperammonemic encephalopathy received continuous venovenous hemofiltration (CVVH) or peritoneal dialysis (PD). Although the hyperammonemia was relieved more effectively in the six patients on CVVH than the seven on PD, most of these patients suffered from severe neurologic deficits. Recurrent hyperammonemic episodes (7 pts, 35%), recurrent and reversible acute hepatic dysfunction (5 pts, 25%), and focal cerebral infarction (2 pts, 10%) were noted. The neonates with hyperammonemic encephalopathy had extensive brain injuries at the onset of hyperammonemia, followed by encephalomalacia and brain atrophy at quite an early age. Genetic testing for the ASS1 gene revealed a different mutation spectrum from those of other ethnicities; Three common mutations, c.421-2A>G (37.8%), c.1128-6_1188dup67 (18.9%), and p.Gly324Ser (16.2%), accounted for 73% of the mutations. The poor outcome was expected in patients with the peak ammonia level at onset over 600μmol/L, whose proportion was higher in the neonatal presentation group than in the presymptomatic/late presentation group. Our findings add to the current understanding of the ethnic diversity of CTLN1 from both clinical and genetic perspectives.

// In Ah Park 1, * , Sun-Hee Heo 1, 2, * , In Hye Song 1 , Young-Ae Kim 1, 2 , Hye Seon Park 1, 2 , Won Seon Bang 1, 2 , Suk Young Park 1, 2 , Jeong-Hyon Jo 3 , Hee Jin Lee 1, ** , Gyungyub Gong 1, ** 1 Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea 2 Asan Center for Cancer Genome Discovery, Asan Institute for Life Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea 3 Department of Pathology, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea * Authors share co-first authorship ** Authors share co-corresponding authorship Correspondence to: Hee Jin Lee, email: backlila@gmail.com Gyungyub Gong, email: gygong@amc.seoul.kr Keywords: breast carcinoma, tumor-infiltrating lymphocytes, endoplasmic reticulum, high-mobility group protein Received: April 23, 2016 Accepted: July 19, 2016 Published: August 2, 2016 ABSTRACT Background: Although the prognostic and predictive significance of tumor-infiltrating lymphocytes (TILs) in triple-negative breast cancer (TNBC) have been shown, the cause of the TIL influx is unclear. Here, we investigated whether extracellular secretion of HMGN1 is associated with TIL influx, as well as increased endoplasmic reticulum stress (ERS), in human TNBC. Methods: We reviewed the slides of 767 patients with TNBC and evaluated the TIL levels. We also assessed the expression of HMGs and several ERS-associated molecules using immunohistochemical staining. Western blot analysis of human TNBC cell lines and pharmacological ERS inducers was used to determine if HMGN1 migrates from the nucleus to the extracellular space in response to ERS. Results: On immunohistochemical staining, either higher nuclear or cytoplasmic expression of both HMGB1 and HMGN1 was significantly associated with ERS. TILs showed a positive correlation with the cytoplasmic expression of the HMGs. Western blot analysis of TNBC cell lines showed that ERS induction resulted in the secretion of HMG proteins. Conclusions: This is the first study to elucidate the associations among ERS, secretion of HMGs, and degree of TILs in TNBCs. Understanding the mechanisms of TIL influx will help in the development of effective immunotherapeutic agents for TNBC.

Periodontitis is an inflammatory disease, which, when severe, can result in tooth loss, that affects the quality of life. S100A2 was previously identified as a component of gingival crevicular fluid (GCF) via proteome analysis, but it has not been investigated whether S100A2 plays a role in periodontitis. In this study, we analyzed mRNA expression of S100A2 in gingival tissues from normal and classified periodontal disease patients and compared it to that of S100A8 and S100A9. Quantitative real time-PCR revealed that the mRNA expression levels of S100A2, S100A8, and S100A9 were significantly upregulated in gingival tissues with gingivitis, moderate periodontitis, and severe periodontitis compared to normal tissues. In addition, S100A2 proteins in GCF and the conditioned media of lipopolysaccharide (LPS)-treated Jurkat cells were confirmed by ELISA. S100A2 protein levels were significantly higher in GCF in gingivitis and moderate periodontitis groups than in normal groups. S100A2 mRNA expression and protein secretion were also increased by LPS stimulation. Based on the up-regulation of S100A2 in LPS-stimulated immune cells, gingival tissues and GCF from periodontal disease groups, we conclude that S100A2 is a functional component in the immune response during periodontitis and may serve as a potential biomarker for periodontitis.

Periodontitis is a very prevalent disease. Therefore, biomarkers for the early and standard diagnoses of periodontitis are urgently needed. TACE is a membrane-bound metalloprotease. Although a recent study suggested that TACE levels in GCF are elevated during periodontal disease, the levels of TACE in GCF at different stages of chronic periodontitis have not been determined. Here, we analyzed the protein levels of TACE in GCF from periodontal disease subjects and confirmed that the protein levels of TACE were higher in the moderate periodontitis groups. TACE is known to be a NF-κB ligand that stimulates RANKL secretion in osteoblasts. To understand the effects of TACE on RANKL and OPG in osteoblasts, we treated MG63 cells with TACE. We observed an increase in RANKL protein expression but a decrease in OPG protein expression. Our data suggest that TACE can induce RANKL expression and promote osteoclastogenesis, thus worsening the outcome of periodontitis. [BMB reports 2011; 44(7): 473-477]

Background: The level of tumor-infiltrating lymphocytes (TILs) and the presence of tertiary lymphoid structures (TLSs) are significant prognostic and predictive factors in primary breast cancer. However, understanding about the differences of TILs and TLSs among different metastatic sites or between primary breast tumors and metastatic sites is limited. Methods: A total of 337 cases of metastatic breast cancer with available hematoxylin and eosin slides of metastatic sites (biopsy, n=185; operation, n=152) were included. We analyzed the percentage of TILs (defined as the percentage of invasive carcinoma stroma infiltrated by lymphocytes in 10% increments; if less than 10% of stroma was infiltrated by TILs, 1% or 5% criteria were used; all available full sections were evaluated) and the presence of TLSs (defined as the lymphoid aggregation with high endothelial venules) in primary and metastatic sites. Results: The mean percentage of TILs in the lung (19.5%) was significantly higher than those in the liver (4.2%), brain (8.3%), and ovary (3.4%, p Conclusion:. Metastatic breast tumors in the lung had more TILs than other sites and matched primary tumors. Underlying mechanism of differences of TILs among metastatic sites should be investigated. Citation Format: Miseon Lee, Hee Jin Lee, In Hye Song, In Ah Park, Sun-Hee Heo, Young-Ae Kim, Won Seon Bang, AReum Lee, Hyeonjin Lee, Heejae Lee, Gyungyub Gong. Comparison of tumor-infiltrating lymphocytes between primary and metastatic tumors in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 697.

Repeated exposure to welding fumes promotes a reversible increase in pulmonary disease risk, but the molecular mechanisms by which welding fumes induce lung injury and how the lung recovers from such insults are unclear. In the present study, pulmonary function and gene-expression profiles in the lung were analyzed by Affymetrix GeneChip microarray after 30 days of consecutive exposure to manual metal arc welding combined with stainless-steel (MMA-SS) welding fumes, and again after 30 days of recovery from MMA-SS fume exposure. In total, 577 genes were identified as being either up-regulated or down-regulated (over twofold changes, p0.05) in the lungs of low-dose or high-dose groups. Differentially expressed genes were classified based on a k-means clustering algorithm and biological functions and molecular networks were further analyzed using Ingenuity Pathways Analysis. Among the genes affected by exposure to or recovery from MMA-SS fumes, the transcriptional changes of 13 genes that were highly altered by treatment were confirmed by quantitative real-time PCR. Notably, Mmp12, Cd5l, Ccl7, Cxcl5, and Spp1 related to the immune response were up-regulated only in the exposure group, whereas Trem2, IgG-2a, Igh-1a, and Igh were persistently up-regulated in both the exposure and recovery groups. In addition, several genes that might play a role in the repair process of the lung were up-regulated exclusively in the recovery group. Collectively, these data may help elucidate the molecular mechanism of the recovery process of the lung after welding fume exposure.

Background: Gaucher disease (GD) is caused by a deficiency in the lysosomal enzyme glucocerebrosidase. Enzyme replacement therapy (ERT) is recommended for clinical improvement.

Tumours with a high mutation burden exhibit considerable neoantigens and tumour-infiltrating lymphocytes. RNA editing by ADAR1 is a source of changes in epitope. However, ADAR1 expression in cancer cells and tumour-infiltrating lymphocyte levels in triple-negative breast cancer have not been well evaluated. We immunohistochemically examined ADAR1 expression in 681 triple-negative breast cancer patients and analysed their clinicopathological characteristics. We also analysed basal-like tumours using The Cancer Genome Atlas data. Among the 681 triple-negative breast cancer patients, 45.8% demonstrated high ADAR1 expression. Tumours with high ADAR1 expression exhibited high tumour-infiltrating lymphocyte levels, considerable CD8 + T lymphocyte infiltration, high histological grade and high expression of interferon-related proteins, including HLA-ABC, MxA and PKR. Among patients with lymph node metastasis, those with high tumour-infiltrating lymphocyte levels and low ADAR1 expression demonstrated the best disease-free survival. The Cancer Genome Atlas data analysis of basal-like tumours revealed significant positive correlation between ADAR1 and CD8B expression and positive association of high ADAR1 expression with immune responses and apoptosis pathways. We detected high ADAR1 expression in half of the triple-negative breast cancer patients. In addition to DNA mutations, RNA editing can be related to neoantigens; hence, we need to explore non-synonymous mutations exclusively found using RNA sequencing data to identify clinically relevant neoantigens.

This study was undertaken to identify growth hormone (GH) responsive proteins and protein expression patterns by short-term recombinant human growth hormone (rhGH) therapy in patients with idiopathic short stature (ISS) using proteomic analysis. Seventeen children (14 males and three females) with ISS were included. They were treated with rhGH at a dose of 0.31 ± 0.078 mg/kg/week for 3 months. Immunodepletion of six highly-abundant serum proteins followed by 2D DIGE analysis, and subsequent MALDI TOF MS, were employed to generate a panel of proteins differentially expressed after short-term rhGH therapy and verify the differences in serum levels of specific proteins by rhGH therapy. Fourteen spots were differentially expressed after rhGH treatment. Among them, apo E and apo L-1 expression were consistently enhanced, whereas serum amyloid A was reduced after rhGH therapy. The differential expressions of these proteins were subsequently verified by Western blot analysis using sera of the before and after rhGH treatment. This study suggests that rhGH therapy influences lipoprotein metabolism and enhances apo L-1 protein expression in ISS patients.

Background The inflammatory disease periodontitis results in tooth loss and can even lead to diseases of the whole body if not treated. Gingival crevicular fluid (GCF) reflects the condition of the gingiva and contains proteins transuded from serum or cells at inflamed sites. In this study, we aimed to discover potential protein biomarkers for periodontitis in GCF proteome using LC-MS/MS. Results We identified 305 proteins from GCF of healthy individuals and periodontitis patients collected using a sterile gel loading tip by ESI-MS/MS coupled to nano-LC. Among these proteins, about 45 proteins were differentially expressed in the GCF proteome of moderate periodontitis patients when compared to the healthy individuals. We first identified azurocidin in the GCF, but not the saliva, as an upregulated protein in the periodontitis patients and verified its increased expression during periodontitis by ELISA using the GCF of the classified periodontitis patients compared to the healthy individuals. In addition, we found that azurocidin inhibited the differentiation of bone marrow-derived macrophages to osteoclasts. Conclusions Our results show that GCF collection using a gel loading tip and subsequent LC-MS/MS analysis following 1D-PAGE proteomic separation are effective for the analysis of the GCF proteome. Our current results also suggest that azurocidin could be a potential biomarker candidate for the early detection of inflammatory periodontal destruction by gingivitis and some chronic periodontitis. Our data also suggest that azurocidin may have an inhibitory role in osteoclast differentiation and, thus, a protective role in alveolar bone loss during the early stages of periodontitis.

Wilson's disease (WD) is characterized by excessive accumulation of intracellular copper in liver and extrahepatic tissues, leading to significant oxidative stress and tissue damage. To date, several diagnostic biomarkers for WD such as serum ceruloplasmin, serum or urine copper levels and copper content in liver have been identified. However, these biomarkers may not be convincing for the diagnosis in some WD patients. To identify additional novel diagnostic biomarkers, we compared the serum protein profiles of asymptomatic childhood WD patients (n=20), without neurologic manifestation or liver cirrhosis, with normal controls (n=13). Fourteen spots, five up-regulated and nine down-regulated (>2-fold), were differentially expressed in WD patients in comparison to normal control on 2-DE. Among them, three spots were down-regulated in both male and female WD. MS/MS analysis revealed that the three spots were complement component C3, complement factor B and alpha-2 macroglobulin. By comparative proteome analysis, complement component C3, complement factor B and alpha-2 macroglobulin, which are related to oxidative stress and inflammation, turned out to be good candidates for novel diagnostic biomarkers for early stages of WD.

Methods This study included 62 transient or permanent CH patients with normal-sized or enlarged eutopic thyroid. All coding exons of DUOX2 and their intronic flanking sequences were amplified by PCR, and directly sequenced. As for novel sequence variant of DUOX2, functional studies were performed by measuring H2O2 generation in vitro. Clinical presentation was retrospectively reviewed based on medical records.