CTNS Molecular Genetics Profile in a Portuguese Cystinosis Population

Background: Cystinosis is a multisystemic autosomal recessive deficiency of the lysosomal membrane transporter protein (cystinosin) caused by mutations in CTNS gene. Objective: This study summarizes the Portuguese experience in the diagnosis and management of patients with this rare disease over the past few years and reports recurrent mutations in the CTNS gene. Methods: Unrelated patients from different pediatric and adult hospitals all over Portugal with non-nephrotic proteinuria, hypercalciuria, hypokalemia impaired proximal reabsorption of amino acids, glycosuria and hypophosphatemia, suggestive of a Fanconi syndrome and ocular problems, were studied. Intra-leukocyte cystine levels were determined and molecular analysis was performed, to determine the presence or absence of the 57-kb deletion in CTNS, followed by direct sequencing of the coding exons of CTNS. Results: From 1998 to 2017, twenty-one cystinotic patients were biochemically diagnosed. From the remaining seventeen (four deceased), eleven were studied for CTNS gene. Five out of eleven patients were homozygous for the 57-kb deletion (10/22;45.5%), and other five were compound heterozygous for this variant (15/22;68.2%). The other mutations found were p.Q128X (c.721 C>T;2/22), p.S139F (c.755 C>T;4/22) and c.18-21delGACT (p.T7FfsX7;1/22). All of these seventeen cystinotic patients are in treatment. Approximately 84% are adults, 16% are young children, and 54.5% are kidney transplant recipient. Conclusions: The authors would like to emphasize the importance of first screening for the 57-kb deletion since it is very common in our population. This genetic study is the first in our country and it could be the basis for future genetic counseling in Portuguese population.

Relationship between Circulating Plasma Galectin-3 Levels and T-Cell Activation during Cervical Cancer Chemotherapy

Objective: Despite the existence of several therapeutic strategies, the management of cervical cancer remains challenging. Our region has very little data on the interaction between the immune system and the clinical response to chemotherapy. This work examines plasma levels of galectin-3 (Gal-3) and percentages of activated T cells in patients with cervical cancer treated with chemotherapy and investigates if there is a relationship between the rates of these two elements. Methods: We compared data from 37 patients with cervical cancer undergoing chemotherapy and 42 controls with normal cervical cytology. Plasma Gal-3 concentrations were assessed by ELISA and expression of activation markers by T cells (CD69 and HLA-DR) was assessed by flow cytometry at three different time points during chemotherapy. Results: Our results showed that patients had a significantly higher concentration of Gal-3 compared to controls (4.025 vs. 1.340, p 0.001), similarly, they had a significantly high percentage of activated lymphocytes (2.610 vs. 0.731;p 0.0001). According to the response to treatment, patients with no response to treatment had a lower concentration of circulating Gal-3 but had approximately the same percentage of activated CD4 and CD8 lymphocytes as patients with a partial or total response. In addition, we found a positive correlation between the Gal-3 level and CD4 T cells expressing the activation marker CD69 (p 0.05;rho = 0.44). Conclusion: In conclusion, our results show that there would be a relationship between circulating galectin-3 and the percentage of peripheral CD4+CD69+ cells in cervical cancer.

MicroRNA signature in patients with hepatocellular carcinoma associated with type 2 diabetes

BACKGROUND Nonalcoholic steatohepatitis-related cirrhosis is one of the liver complications in type 2 diabetes mellitus(T2DM)and reported to be a risk factor for developing hepatocellular carcinoma(HCC).A reliable screening biomarker of liver cirrhosis(LC)and HCC among T2DM patients is important to reduce the morbidity and mortality of this disease.MicroRNA(miRNA)is considered a key player in HCC and T2DM,and it might be a hidden culprit in diabetes-associated HCC,making it a promising reliable prognostic tool.AIM To investigate the signature of serum miRNAs as early biomarkers for the screening of HCC among diabetic patients.METHODS Expression profiles of miRNAs in serum samples of diabetic LC and diabetic HCC patients were assessed using Illumina sequencing;then,RT-qPCR was used to validate significantly altered miRNAs between the two groups.Candidate miRNAs were tested in serum samples of 200 T2DM patients,270 LC patients,200 HCC patients,and 225 healthy control subjects.Additionally,receiver operating characteristic(ROC)analysis,with area under the curve(AUC),was performed to assess the diagnostic performance of the screened miRNAs for discriminating HCC from LC and nonmalignant patients(LC+T2DM).RESULTS Expression of the sequenced miRNAs in serum was different in HCC vs LCpositive T2DM patients.Two miRNAs(miR-34a,miR-221)were significantly upregulated and five miRNAs(miR-16,miR-23-3p,miR-122-5p,miR-198,miR-199a-3p)were significantly down-regulated in HCC compared to LC patients.Analysis of ROC curve demonstrated that the combination of these seven miRNAs can be used as a reliable biomarker for detection of HCC in diabetic patients,as it could identify HCC with high diagnostic accuracy in diabetic LC patients(AUC=0.993)and in diabetic nonmalignant patients(AUC=0.961).CONCLUSION This study validates a panel of serum miRNAs that can be used as a reliable noninvasive screening biomarker of HCC among T2DM cirrhotic and noncirrhotic patients.The study recommends further research to shed light on a possible role of c-Met in T2DM-associated HCC via the miRNA regulatory pathway.

Prenatal exposure to the fluoride containing psychiatric drug fluoxetine and anti-oxidative alterations in the neonatal rat brain

Fluoride is a key ingredient of many psychiatric drugs like fluoxetine(Prozac■Fluoxetine■).Pregnant women frequently use this drug as they suffer from depression and anxiety disorders during this period.Fluoxetine is able to reach the fetus through the placenta and passes to the newborn through milk.In the present study,female Wistar rats were treated with 5,10,and 20 mg/L fluoxetine(containing 94% fluorides)from pregnancy day 10 to day 20.After delivery,the levels of the enzymatic antioxidants in the brain of their offspring at postnatal day 2 were measured.The results showed that,in all fluoxetine exposed groups compared with the control group,there was a significant decrease(P<0.01)in the glutathione,catalase,glutathione S-transferases and potassium and a non-significant increase(P>0.05)in the activity of malondialdehyde and creatine kinase.The results suggest that fluoxetine may be a developmental neurotoxicant due to presence of fluoride hence must be used carefully during pregnancy.

Virological course of hepatitis A virus as determined by real time RT-PCR: Correlation with biochemical, immunological and genotypic profiles

AIM： To undertake analysis of hepatitis A viral load, alanine aminotransferase （ALT）, and viral genotypes with duration of viremia, and to correlate these parameters with CD4^＋/ CD8^＋ lymphocyte populations that control cell-mediated immunity. METHODS： Cell counts were carried out using fresh whole blood collected in EDTA vials using a fluorescence activated cell sorter. Hepatitis A virus （HAV） RNA was extracted from blood serum, reverse transcribed into cDNA and quantified by Real-Time polymerase chain reaction and was genotyped. RESULTS： Among 11 patients, 10 could be analyzed completely. Of these, 3 had severe acute hepatitis （s-AH） and the remainder had a self-limited acute hepatitis A （AHA）, with one patient with fulminant disease （encephalopathy Grade IV） dying on the 4^th d. The ALT level was significantly higher both in AHA （1070.9±894.3; P = 0.0014） and s-AH （1713.9±886.3; P = 0.001） compared to normal controls （23.6±7.2）. The prothrombin time in s-AH patients （21.0 ±2.0; P=0.02） was significantly higher than in AHA （14.3±1.1;P = 0.44）. The CD4^＋/CD8^＋ ratio in AHA patients （1.17 ＋ 0.11; P = 0.22） and s-AH （0.83 ＋ 0.12; P = 0.0002） were lower than seen in normal healthy controls （1.52）. Self-limited cases had peak viral load at the beginning of analysis while in s-AH patients this occurred at the 15TM or 30^th d. In acute and severe groups, one patient each belonged to genotype IA, with the remaining 8 cases belonging to genotype IIIA. The only fulminant hepatic failure case belonged to genotype IA. HAV viral load and AIT values collected during the entire course of the selflimited infection were directly correlated but this was not the case for s-AH patients.CONCLUSION： Based on a small-scale study, the persistently higher viral load of s-AH might be due to diminished cellular immunity and hemolysis. The duration of viremia was dependent on the host, as the viral genotype had no apparent role in clinical outcome of AVH and s-AH cases.

Candidate colorectal cancer predisposing gene variants in Chinese early-onset and familial cases

AIM: To investigate whether whole-exome sequencing may serve as an efficient method to identify known or novel colorectal cancer(CRC) predisposing genes in early-onset or familial CRC cases.METHODS: We performed whole-exome sequencing in 23 Chinese patients from 21 families with nonpolyposis CRC diagnosed at ≤ 40 years of age, or from multiple affected CRC families with at least 1 firstdegree relative diagnosed with CRC at ≤ 55 years of age.Genomic DNA from blood was enriched for exome sequences using the Sure Select Human All Exon Kit, version 2(Agilent Technologies) and sequencing was performed on an Illumina Hi Seq 2000 platform.Data were processed through an analytical pipeline to search for rare germline variants in known or novel CRC predisposing genes.RESULTS: In total, 32 germline variants in 23 genes were identified and confirmed by Sanger sequencing.In 6 of the 21 families(29%), we identified 7 mutations in 3 known CRC predisposing genes including MLH1(5 patients), MSH2(1 patient), and MUTYH(biallelic, 1 patient), five of which were reported as pathogenic.Inthe remaining 15 families, we identified 20 rare and novel potentially deleterious variants in 19 genes, six of which were truncating mutations.One previously unreported variant identified in a conserved region of EIF2AK4(p.Glu738_Asp739insA rgA rg) was found to represent a local Chinese variant, which was significantly enriched in our early-onset CRC patient cohort compared to a control cohort of 100 healthy Chinese individuals scored negative by colonoscopy(33.3% vs 7%, P < 0.001).CONCLUSION: Whole-exome sequencing of early-onset or familial CRC cases serves as an efficient method to identify known and potential pathogenic variants in established and novel candidate CRC predisposing genes.

Cytotoxicity and Selectivity in Skin Cancer by SapC-DOPS Nanovesicles

Squamous cell carcinoma (SCC) and melanoma are malignant human cancers of the skin with an annual mortality that exceeds 10,000 cases every year in the USA alone. In this study, the lysosomal protein saposin C (SapC) and the phospholipid dioloylphosphatidylserine (DOPS) were assembled into cancer-selective nanovesicles (SapC-DOPS) and successfully tested using several in vitro and in vivo skin cancer models. Using MTT assay that measures the percentage of cell death, SapC-DOPS cytotoxic effect on three skin tumor cell lines (squamous cell carcinoma, SK-MEL-28, and MeWo) was compared to two normal nontumorigenic skin cells lines, normal immortalized keratinocyte (NIK) and human fibroblast cell (HFC). We observed that the nanovesicles selectively killed the skin cancer cells by inducing apoptotic cell death whereas untransformed skin cancer cells remained unaffected. Using subcutaneous skin tumor xenografts, animals treated with SapC-DOPS by subcutaneous injection showed a 79.4% by volume tumor reduced compared to the control after 4 days of treatment. We observed that the nanovesicles killed skin cancer cells by inducing apoptotic cell death compared to the control as revealed by TUNEL staining of xenograft tumor sections.

Identification of a high frequency of chromosomal rearrangements in the centromeric regions of prostate cancer patients

The aim of the present investigation was to study the major chromosomal aberrations （CA） like deletion, translocation, inversion and mosaic in prostate cancer patients of Tamilnadu, Southern India. Totally 45 blood samples were collected from various hospitals in Tamilnadu, Southern India. Equal numbers of normal healthy subjects were chosen after signing a consent form. Volunteers provided blood samples （5 ml） to establish leukocyte cultures. Cytogenetic studies were performed by using Giemsaanding technique and finally the results were ensured by spectral karyotyping （SKY） technique. In the present investigation, major CA like deletion, translocation, inversion and mosaic were identifed in experimental subjects. Results showed frequent CA in chromosomes 1, 3, 5, 6, 7, 9, 13, 16, 18 and X. In comparison with experimental subjects, the control subjects exhibited very low levels of major CA （P〈0.05）. In the present study, the high frequency of centromeric rearrangements indicates a potential role for mitotic irregularities associated with the centromere in prostate cancer tumorigenesis. Identification of chromosome alterations may be helpful in understanding the molecular basis of the disease in better manner.

Molecular Dynamics Simulations of DOPC Lipid Bilayers: The Effect of Lennard-Jones Parameters of Hydrocarbon Chains

The current Chemistry at Harvard Molecular Mechanics (CHARMM) force field cannot accurately describe the properties of unsaturated phospholipid membranes. In this paper, a series of simulations was performed in which the Lennard- Jones (L-J) parameters of lipid acyl chains of dioleoylphosphatidylcholine (DOPC) were systematically adjusted. The results showed that adjustment of the L-J parameters in lipid acyl chains can significantly improve the current CHARMM force field. It was found that the L-J parameters have different influences on the order parameters of the top half and bottom half of the chain, separated by the cis double bond. The order parameters of the top half and the bottom half of the chain are related to the area/lipid and the length of the chain, respectively.

Keratinocytes costimulate naive human T cells via CD2: a potential target to prevent the development of proinflammatory Th1 cells in the skin

The interplay between keratinocytes and immune cells,especially T cells,plays an important role in the pathogenesis of chronic inflammatory skin diseases.During psoriasis,keratinocytes attract T cells by releasing chemokines,while skin-infiltrating selfreactive T cells secrete proinflammatory cytokines,e.g.,IFN γand IL-17A,that cause epidermal hyperplasia.Similarly,in chronic graftversus-host disease,allogenic IFN γ-producing Th1/Tc1 and IL-17-producing Th17/Tc17 cells are recruited by keratinocyte-derived chemokines and accumulate in the skin.However,whether keratinocytes act as nonprofessional antigen-presenting cells to directly activate naive human T cells in the epidermis remains unknown.Here,we demonstrate that under proinflammatory conditions,primary human keratinocytes indeed activate naive human T cells.This activation required cell contact and costimulatory signaling via CD58/CD2 and CD54/LFA-1.Naive T cells costimulated by keratinocytes selectively differentiated into Th1 and Th17 cells.In particular,keratinocyte-initiated Th1 differentiation was dependent on costimulation through CD58/CD2.The latter molecule initiated STAT1 signaling and IFN γproduction in T cells.Costimulation of T cells by keratinocytes resulting in Th1 and Th17 differentiation represents a new explanation for the local enrichment of Th1 and Th17 cells in the skin of patients with a chronic inflammatory skin disease.Consequently,local interference with T cell–keratinocyte interactions may represent a novel strategy for the treatment of Th1 and Th17 cell-driven skin diseases.

Human Genetic Variants Associated with COVID-19 Severity are Enriched in Immune and Epithelium Regulatory Networks

Human genetic variants can influence the severity of symptoms infected with SARS-COV-2.Several genome-wide asso-ciation studies have identified human genomic risk single nucleotide polymorphisms(SNPs)associated with coronavirus disease 2019(COVID-19)severity.However,the causal tissues or cell types underlying COVID-19 severity are uncertain.In addition,candidate genes associated with these risk SNPs were investigated based on genomic proximity instead of their functional cellular contexts.Here,we compiled regulatory networks of 77 human contexts and revealed those risk SNPs’enriched cellular contexts and associated risk SNPs with transcription factors,regulatory elements,and target genes.Twenty-one human contexts were identified and grouped into two categories:immune cells and epithelium cells.We further aggregated the regulatory networks of immune cells and epithelium cells.These two aggregated regulatory networks were investigated to reveal their association with risk SNPs’regulation.Two genomic clusters,the chemokine receptors cluster and the oligoadenylate synthetase(OAS)cluster,showed the strongest association with COVID-19 severity,and they had different regulatory programs in immune and epithelium contexts.Our findings were supported by analysis of both SNP array and whole genome sequencing-based genome wide association study(GWAS)summary statistics.

单基因遗传性高血压:一类可“治愈”的高血压

Hypertension is a global problem that affects more than 1 billion people worldwide with the increased prevalence year by year[1,2].It contributes to major impacts on health including morbidity and all-cause mortality,as well as consumption of substantial health care expenses.Understanding the complex pathophysiology and risk factors involved in the development of elevated blood pressure can help treat the disease to better prevent life-threatening conditions and alleviate the socio-economic burden.The hereditary nature of hypertension relies on that up to 30%of blood pressure variation is due to genetics and an individual’s genetic predisposition to hypertensive disease ranges from 15%to 35%[3].

Mutation analysis and prenatal diagnosis of a family with Griscelli syndrome type 2: two novel mutations in the RAB27A gene

Griscelli syndrome type 2 (GS2;OMIM#607624) is a rare autosomal recessive disorder characterized by hypomelanosis with immunologic abnormalities and haemophagocytic lymphohistocytosis.[1] Neurological manifestations were reported in 67% of GS2 patients.[2]It is caused by mutations in the RAB27A gene.[3] The RAB27A gene encodes Rab27a,a member of the small GTPase superfamily,involved in vesicular fusion and trafficking.[3] Mutations in the MYO5A,RAB27A,or MLPH genes cause GS 1,GS2 or GS3,respectively.It has been demonstrated that the tripartite protein complex (Rab27a/melanophilin/myosin Va) in melanocytes is needed for capturing mature melanosomes for transferring to keratinocytes.