Publications by authors named "Sampathkumar Rangasamy"

33 Publications

Improved methods for RNAseq-based alternative splicing analysis.

Sci Rep 2021 05 24;11(1):10740. Epub 2021 May 24.

Quantitative Medicine and Systems Biology Division, Translational Genomics Research Institute, Phoenix, AZ, USA.

The robust detection of disease-associated splice events from RNAseq data is challenging due to the potential confounding effect of gene expression levels and the often limited number of patients with relevant RNAseq data. Here we present a novel statistical approach to splicing outlier detection and differential splicing analysis. Our approach tests for differences in the percentages of sequence reads representing local splice events. We describe a software package called Bisbee which can predict the protein-level effect of splice alterations, a key feature lacking in many other splicing analysis resources. We leverage Bisbee's prediction of protein level effects as a benchmark of its capabilities using matched sets of RNAseq and mass spectrometry data from normal tissues. Bisbee exhibits improved sensitivity and specificity over existing approaches and can be used to identify tissue-specific splice variants whose protein-level expression can be confirmed by mass spectrometry. We also applied Bisbee to assess evidence for a pathogenic splicing variant contributing to a rare disease and to identify tumor-specific splice isoforms associated with an oncogenic mutation. Bisbee was able to rediscover previously validated results in both of these cases and also identify common tumor-associated splice isoforms replicated in two independent melanoma datasets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-89938-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8144374PMC
May 2021

Congenital myasthenic syndrome caused by a frameshift insertion mutation in .

Neurol Genet 2020 Aug 30;6(4):e468. Epub 2020 Jun 30.

theNeurogenomics Division (S.S., J.K., K.R., N.B., C.B., A.L.S., M.R., R.R., M.D.B., A.M.C., M.J.H, V.N., S.R.), Translational Genomics Research Institute, Center for Rare Childhood Disorders, Phoenix, AZ; Fulgent Genetics (S.P.S.), Temple City, CA; Department of Neurology (P.B.S.), University of California Los Angeles; David Geffen School of Medicine (P.B.S.), Los Angeles; Department of Pathology and Laboratory Medicine (H.L., S.F.N.), University of California, Los Angeles; Department of Human Genetics (H.L., S.F.N.), David Geffen School of Medicine; Department of Neurology (I.S.), Columbia University, Center for Statistical Genetics, New York; Department of Translational Genomics (D.W.C.), University of Southern California, Los Angeles; Providence Sacred Heart Medical Center and Children's Hospital (S.P.Y.), Spokane, WA; Department of Pathology (S.A.M), University of Iowa, Carver College of Medicine; and Neuromuscular Clinic and Research Center (K.S.), Phoenix, AZ.

Objective: Description of a new variant of the glutamine-fructose-6-phosphate transaminase 1 () gene causing congenital myasthenic syndrome (CMS) in 3 children from 2 unrelated families.

Methods: Muscle biopsies, EMG, and whole-exome sequencing were performed.

Results: All 3 patients presented with congenital hypotonia, muscle weakness, respiratory insufficiency, head lag, areflexia, and gastrointestinal dysfunction. Genetic analysis identified a homozygous frameshift insertion in the gene (NM_001244710.1: c.686dupC; p.Arg230Ter) that was shared by all 3 patients. In one of the patients, inheritance of the variant was through uniparental disomy (UPD) with maternal origin. Repetitive nerve stimulation and single-fiber EMG was consistent with the clinical diagnosis of CMS with a postjunctional defect. Ultrastructural evaluation of the muscle biopsy from one of the patients showed extremely attenuated postsynaptic folds at neuromuscular junctions and extensive autophagic vacuolar pathology.

Conclusions: These results expand on the spectrum of known loss-of-function mutations in CMS12 and in one family demonstrate a novel mode of inheritance due to UPD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1212/NXG.0000000000000468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357421PMC
August 2020

Transcriptomics analysis of pericytes from retinas of diabetic animals reveals novel genes and molecular pathways relevant to blood-retinal barrier alterations in diabetic retinopathy.

Exp Eye Res 2020 06 4;195:108043. Epub 2020 May 4.

Surgery/Ophthalmology, USA; Cell Biology & Physiology, University of New Mexico, Albuquerque, NM, USA; NMVA Health Care System, Albuquerque, NM, USA. Electronic address:

Selective pericyte loss, the histological hallmark of early diabetic retinopathy (DR), enhances the breakdown of the blood-retinal barrier (BRB) in diabetes. However, the role of pericytes on BRB alteration in diabetes and the signaling pathways involved in their effects are currently unknown. To understand the role of diabetes-induced molecular alteration of pericytes, we performed transcriptomic analysis of sorted retinal pericytes from mice model of diabetes. Retinal tissue from non-diabetic and diabetic (duration 3 months) mouse eyes (n = 10 in each group) were used to isolate pericytes through fluorescent activated cell sorting (FACS) using pericyte specific fluorescent antibodies, PDGFRb-APC. For RNA sequencing and qPCR analysis, a cDNA library was generated using template switching oligo and the resulting libraries were sequenced using paired-end Illumina sequencing. Molecular functional pathways were analyzed using differentially expressed genes (DEGs). Differential expression analysis revealed 217 genes significantly upregulated and 495 genes downregulated, in pericytes isolated from diabetic animals. These analyses revealed a core set of differentially expressed genes that could potentially contribute to the pericyte dysfunction in diabetes and highlighted the pattern of functional connectivity between key candidate genes and blood retinal barrier alteration mechanisms. The top up-regulated gene list included: Ext2, B3gat3, Gpc6, Pip5k1c and Pten and down-regulated genes included: Notch3, Xbp1, Gpc4, Atp1a2 and AKT3. Out of these genes, we further validated one of the down regulated genes, Notch 3 and its role in BRB alteration in diabetic retinopathy. We confirmed the downregulation of Notch3 expression in human retinal pericytes exposed to Advanced Glycation End-products (AGEs) treatment mimicking the chronic hyperglycemia effect. Exploration of pericyte-conditioned media demonstrated that loss of NOTCH3 in pericyte led to increased permeability of endothelial cell monolayers. Collectively, we identify a role for NOTCH3 in pericyte dysfunction in diabetes. Further validation of other DEGs to identify cell specific molecular change through whole transcriptomic approach in diabetic retina will provide novel insight into the pathogenesis of DR and novel therapeutic targets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.exer.2020.108043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7323486PMC
June 2020

Do Genomic Factors Play a Role in Diabetic Retinopathy?

J Clin Med 2020 Jan 14;9(1). Epub 2020 Jan 14.

Department of Surgery/Ophthalmology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA.

Although there is strong clinical evidence that the control of blood glucose, blood pressure, and lipid level can prevent and slow down the progression of diabetic retinopathy (DR) as shown by landmark clinical trials, it has been shown that these factors only account for 10% of the risk for developing this disease. This suggests that other factors, such as genetics, may play a role in the development and progression of DR. Clinical evidence shows that some diabetics, despite the long duration of their diabetes (25 years or more) do not show any sign of DR or show minimal non-proliferative diabetic retinopathy (NPDR). Similarly, not all diabetics develop proliferative diabetic retinopathy (PDR). So far, linkage analysis, candidate gene studies, and genome-wide association studies (GWAS) have not produced any statistically significant results. We recently initiated a genomics study, the Diabetic Retinopathy Genetics (DRGen) Study, to examine the contribution of rare and common variants in the development of different phenotypes of DR, as well as their responsiveness to anti-VEGF treatment in diabetic macular edema (DME). Our preliminary findings reveal a novel set of genetic variants involved in the angiogenesis and inflammatory pathways that contribute to DR progression or protection. Further investigation of variants can help to develop novel biomarkers and lead to new therapeutic targets in DR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/jcm9010216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019561PMC
January 2020

Compound heterozygous mutations in SNAP29 is associated with Pelizaeus-Merzbacher-like disorder (PMLD).

Hum Genet 2019 Dec 20;138(11-12):1409-1417. Epub 2019 Nov 20.

Center for Rare Childhood Disorders, Translational Genomics Research Institute, Phoenix, AZ, USA.

Pelizaeus-Merzbacher-like disease (PMLD) is an autosomal recessive hypomyelinating leukodystrophy, which is clinically and radiologically similar to X-linked Pelizaeus-Merzbacher disease (PMD). PMLD is characterized by early-onset nystagmus, delayed development (motor delay, speech delay and dysarthria), dystonia, hypotonia typically evolving into spasticity, ataxia, seizures, optic atrophy, and diffuse leukodystrophy on magnetic resonance imaging (MRI). We identified a 12-year-old Caucasian/Hispanic male with the classical clinical characteristics of PMLD with lack of myelination of the subcortical white matter, and absence of the splenium of corpus callosum. Exome sequencing in the trio revealed novel compound heterozygous pathogenic mutations in SNAP29 (p.Leu119AlafsX15, c.354DupG and p.0?, c.2T > C). Quantitative analysis of the patient's blood cells through RNA sequencing identified a significant decrease in SNAP29 mRNA expression, while western blot analysis on fibroblast cells revealed a lack of protein expression compared to parental and control cells. Mutations in SNAP29 have previously been associated with cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK) syndrome. Typical skin features described in CEDNIK syndrome, such as generalized ichthyosis and keratoderma, were absent in our patient. Moreover, the early onset nystagmus and leukodystrophy were consistent with a PMLD diagnosis. These findings suggest that loss of SNAP29 function, which was previously associated with CEDNIK syndrome, is also associated with PMLD. Overall, our study expands the genetic spectrum of PMLD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00439-019-02077-7DOI Listing
December 2019

Rare De Novo Missense Variants in RNA Helicase DDX6 Cause Intellectual Disability and Dysmorphic Features and Lead to P-Body Defects and RNA Dysregulation.

Am J Hum Genet 2019 09 15;105(3):509-525. Epub 2019 Aug 15.

Laboratoire de Génétique Médicale, Institut de Génétique Médicale d'Alsace, INSERM U1112, Fédération de Médecine Translationnelle de Strasbourg, Université de Strasbourg, 67081 Strasbourg, France; Molecular Genetics Unit, Strasbourg University Hospital, 67000 Strasbourg, France.

The human RNA helicase DDX6 is an essential component of membrane-less organelles called processing bodies (PBs). PBs are involved in mRNA metabolic processes including translational repression via coordinated storage of mRNAs. Previous studies in human cell lines have implicated altered DDX6 in molecular and cellular dysfunction, but clinical consequences and pathogenesis in humans have yet to be described. Here, we report the identification of five rare de novo missense variants in DDX6 in probands presenting with intellectual disability, developmental delay, and similar dysmorphic features including telecanthus, epicanthus, arched eyebrows, and low-set ears. All five missense variants (p.His372Arg, p.Arg373Gln, p.Cys390Arg, p.Thr391Ile, and p.Thr391Pro) are located in two conserved motifs of the RecA-2 domain of DDX6 involved in RNA binding, helicase activity, and protein-partner binding. We use functional studies to demonstrate that the first variants identified (p.Arg373Gln and p.Cys390Arg) cause significant defects in PB assembly in primary fibroblast and model human cell lines. These variants' interactions with several protein partners were also disrupted in immunoprecipitation assays. Further investigation via complementation assays included the additional variants p.Thr391Ile and p.Thr391Pro, both of which, similarly to p.Arg373Gln and p.Cys390Arg, demonstrated significant defects in P-body assembly. Complementing these molecular findings, modeling of the variants on solved protein structures showed distinct spatial clustering near known protein binding regions. Collectively, our clinical and molecular data describe a neurodevelopmental syndrome associated with pathogenic missense variants in DDX6. Additionally, we suggest DDX6 join the DExD/H-box genes DDX3X and DHX30 in an emerging class of neurodevelopmental disorders involving RNA helicases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajhg.2019.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731366PMC
September 2019

Biallelic VARS variants cause developmental encephalopathy with microcephaly that is recapitulated in vars knockout zebrafish.

Nat Commun 2019 02 12;10(1):708. Epub 2019 Feb 12.

Pediatric Department B' Emek Medical Center, Afula, 1834111, Israel.

Aminoacyl tRNA synthetases (ARSs) link specific amino acids with their cognate transfer RNAs in a critical early step of protein translation. Mutations in ARSs have emerged as a cause of recessive, often complex neurological disease traits. Here we report an allelic series consisting of seven novel and two previously reported biallelic variants in valyl-tRNA synthetase (VARS) in ten patients with a developmental encephalopathy with microcephaly, often associated with early-onset epilepsy. In silico, in vitro, and yeast complementation assays demonstrate that the underlying pathomechanism of these mutations is most likely a loss of protein function. Zebrafish modeling accurately recapitulated some of the key neurological disease traits. These results provide both genetic and biological insights into neurodevelopmental disease and pave the way for further in-depth research on ARS related recessive disorders and precision therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-07953-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6372652PMC
February 2019

A novel FBXO28 frameshift mutation in a child with developmental delay, dysmorphic features, and intractable epilepsy: A second gene that may contribute to the 1q41-q42 deletion phenotype.

Am J Med Genet A 2018 07;176(7):1549-1558

Neurogenomics Division, Center for Rare Childhood Disorders (C4RCD), Translational Genomics Research Institute, Phoenix, Arizona.

Chromosome 1q41-q42 deletions have recently been associated with a recognizable neurodevelopmental syndrome of early childhood (OMIM 612530). Within this group, a predominant phenotype of developmental delay (DD), intellectual disability (ID), epilepsy, distinct dysmorphology, and brain anomalies on magnetic resonance imaging/computed tomography has emerged. Previous reports of patients with de novo deletions at 1q41-q42 have led to the identification of an evolving smallest region of overlap which has included several potentially causal genes including DISP1, TP53BP2, and FBXO28. In a recent report, a cohort of patients with de novo mutations in WDR26 was described that shared many of the clinical features originally described in the 1q41-q42 microdeletion syndrome (MDS). Here, we describe a novel germline FBXO28 frameshift mutation in a 3-year-old girl with intractable epilepsy, ID, DD, and other features which overlap those of the 1q41-q42 MDS. Through a familial whole-exome sequencing study, we identified a de novo FBXO28 c.972_973delACinsG (p.Arg325GlufsX3) frameshift mutation in the proband. The frameshift and resulting premature nonsense mutation have not been reported in any genomic database. This child does not have a large 1q41-q42 deletion, nor does she harbor a WDR26 mutation. Our case joins a previously reported patient also in whom FBXO28 was affected but WDR26 was not. These findings support the idea that FBXO28 is a monogenic disease gene and contributes to the complex neurodevelopmental phenotype of the 1q41-q42 gene deletion syndrome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ajmg.a.38712DOI Listing
July 2018

Neonatal epileptic encephalopathy caused by de novo GNAO1 mutation misdiagnosed as atypical Rett syndrome: Cautions in interpretation of genomic test results.

Semin Pediatr Neurol 2018 07 16;26:28-32. Epub 2017 Aug 16.

Center for Rare Childhood Disorders, Translational Genomics Research Institute, Phoenix, AZ; Neurogenomics Division, Translational Genomics Research Institute, Phoenix, AZ; School of Life Sciences, Arizona State University, Tempe, AZ. Electronic address:

Epileptic encephalopathies are childhood brain disorders characterized by a variety of severe epilepsy syndromes that differ by the age of onset and seizure type. Until recently, the cause of many epileptic encephalopathies was unknown. Whole exome or whole genome sequencing has led to the identification of several causal genes in individuals with epileptic encephalopathy, and the list of genes has now expanded greatly. Genetic testing with epilepsy gene panels is now done quite early in the evaluation of children with epilepsy, following brain imaging, electroencephalogram, and metabolic profile. Early infantile epileptic encephalopathy (EIEE1; OMIM #308350) is the earliest of these age-dependent encephalopathies, manifesting as tonic spasms, myoclonic seizures, or partial seizures, with severely abnormal electroencephalogram, often showing a suppression-burst pattern. In this case study, we describe a 33-month-old female child with severe, neonatal onset epileptic encephalopathy. An infantile epilepsy gene panel test revealed 2 novel heterozygous variants in the MECP2 gene; a 70-bp deletion resulting in a frameshift and truncation (p.Lys377ProfsX9) thought to be pathogenic, and a 6-bp in-frame deletion (p.His371_372del), designated as a variant of unknown significance. Based on this test result, the diagnosis of atypical Rett syndrome (RTT) was made. Family-based targeted testing and segregation analysis, however, raised questions about the pathogenicity of these specific MECP2 variants. Whole exome sequencing was performed in this family trio, leading to the discovery of a rare, de novo, missense mutation in GNAO1 (p. Leu284Ser). De novo, heterozygous mutations in GNAO1 have been reported to cause early infantile epileptic encephalopathy-17 (EIEE17; OMIM 615473). The child's severe phenotype, the family history and segregation analysis of variants and prior reports of GNAO1-linked disease allowed us to conclude that the GNAO1 mutation, and not the MECP2 variants, was the cause of this child's neurological disease. With the increased use of genetic panels and whole exome sequencing, we will be confronted with lists of gene variants suspected to be pathogenic or of unknown significance. It is important to integrate clinical information, genetic testing that includes family members and correlates this with the published clinical and scientific literature, to help one arrive at the correct genetic diagnosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.spen.2017.08.008DOI Listing
July 2018

Exploring genome-wide DNA methylation patterns in Aicardi syndrome.

Epigenomics 2017 11 2;9(11):1373-1386. Epub 2017 Oct 2.

Center for Rare Childhood Disorders, Translational Genomics Research Institute, 445 N 5th Street, Phoenix, AZ, USA.

Aim: To explore differential DNA methylation (DNAm) in Aicardi syndrome (AIC), a severe neurodevelopmental disorder with largely unknown etiology.

Patients & Methods: We characterized DNAm in AIC female patients and parents using the Illumina 450 K array. Differential DNAm was assessed using the local outlier factor algorithm, and results were validated via qPCR in a larger set of AIC female patients, parents and unrelated young female controls. Functional epigenetic modules analysis was used to detect pathways integrating both genome-wide DNAm and RNA-seq data.

Results & Conclusion: We detected differential methylation patterns in AIC patients in several neurodevelopmental and/or neuroimmunological networks. These networks may be part of the underlying pathogenic mechanisms involved in the disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2217/epi-2017-0060DOI Listing
November 2017

Case Report: Novel mutations in are associated with autosomal dominant tonic-clonic and myoclonic epilepsy and recessive Parkinsonism, psychosis, and intellectual disability.

F1000Res 2017 24;6:553. Epub 2017 Apr 24.

Center for Rare Childhood Disorders, Translational Genomics Research Institute, Phoenix, AZ, 85004, USA.

Mutations disrupting presynaptic protein TBC1D24 are associated with a variable neurological phenotype, including DOORS syndrome, myoclonic epilepsy, early-infantile epileptic encephalopathy, and non-syndromic hearing loss. In this report, we describe a family segregating autosomal dominant epilepsy, and a 37-year-old Caucasian female with a severe neurological phenotype including epilepsy, Parkinsonism, psychosis, visual and auditory hallucinations, gait ataxia and intellectual disability. Whole exome sequencing revealed two missense mutations in the gene segregating within this family (c.1078C>T; p.Arg360Cys and c.404C>T; p.Pro135Leu). The female proband who presents with a severe neurological phenotype carries both of these mutations in a compound heterozygous state. The p.Pro135Leu variant, however, is present in the proband's mother and sibling as well, and is consistent with an autosomal dominant pattern linked to tonic-clonic and myoclonic epilepsy. In conclusion, we describe a single family in which mutations cause expanded dominant and recessive phenotypes. In addition, we discuss and highlight that some variants in might cause a dominant susceptibility to epilepsy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.12688/f1000research.10588.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5473401PMC
April 2017

Association of increased levels of MCP-1 and cathepsin-D in young onset type 2 diabetes patients (T2DM-Y) with severity of diabetic retinopathy.

J Diabetes Complications 2017 May 10;31(5):804-809. Epub 2017 Mar 10.

Department of Cell and Molecular Biology and Dr. Rema Mohan High-Throughput Screening (HTS) Lab, Madras Diabetes Research Foundation & Dr. Mohan's Diabetes Specialities Centre, Gopalapuram, Chennai 600086, India.. Electronic address:

Aim: Young onset type 2 diabetes patients (T2DM-Y) have been shown to possess an increased risk of developing microvascular complications particularly diabetic retinopathy. However, the molecular mechanisms are not clearly understood. In this study, we investigated the serum levels of monocyte chemotactic protein 1 (MCP-1) and cathepsin-D in patients with T2DM-Y without and with diabetic retinopathy.

Methods: In this case-control study, participants comprised individuals with normal glucose tolerance (NGT=40), patients with type 2 diabetes mellitus (T2DM=35), non-proliferative diabetic retinopathy (NPDR=35) and proliferative diabetic retinopathy (PDR=35). Clinical characterization of the study subjects was done by standard procedures and MCP-1 and cathepsin-D were measured by ELISA.

Results: Compared to control individuals, patients with T2DM-Y, NPDR and PDR exhibited significantly (p<0.001) higher levels of MCP-1. Cathepsin-D levels were also significantly (p<0.001) higher in patients with T2DM-Y without and with diabetic retinopathy. Correlation analysis revealed a positive association (p<0.001) between MCP-1 and cathepsin-D levels. There was also a significant negative correlation of MCP1/cathepsin-D with C-peptide levels. The association of increased levels of MCP-1/cathepsin-D in patients with DR persisted even after adjusting for all the confounding factors.

Conclusion: As both MCP-1 and cathepsin-D are molecular signatures of cellular senescence, we suggest that these biomarkers might be useful to predict the development of retinopathy in T2DM-Y patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jdiacomp.2017.02.017DOI Listing
May 2017

Reduced neuronal size and mTOR pathway activity in the Mecp2 A140V Rett syndrome mouse model.

F1000Res 2016 8;5:2269. Epub 2016 Sep 8.

Neurogenomics Division, Translational Genomics Research Institute, Phoenix, USA; Center for Rare Childhood Disorders, Translational Genomics Research Institute, Phoenix, USA; Barrow Neurological Institute, St.Joseph's Hospital and Medical Center, Phoenix, USA; School of Life Sciences, Arizona State University, Tempe, USA.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutation in the X-linked gene, encoding methyl-CpG-binding protein 2. We have created a mouse model ( A140V "knock-in" mutant) expressing the recurrent human A140V mutation linked to an X-linked mental retardation/Rett syndrome phenotype. Morphological analyses focused on quantifying soma and nucleus size were performed on primary hippocampus and cerebellum granule neuron (CGN) cultures from mutant ( ) and wild type ( ) male mice. Cultured hippocampus and cerebellar granule neurons from mutant animals were significantly smaller than neurons from wild type animals. We also examined soma size in hippocampus neurons from individual female transgenic mice that express both a mutant  (maternal allele) and a wild type gene linked to an eGFP transgene (paternal allele). In cultures from such doubly heterozygous female mice, the size of neurons expressing the mutant (A140V) allele also showed a significant reduction compared to neurons expressing wild type MeCP2, supporting a cell-autonomous role for MeCP2 in neuronal development. IGF-1 (insulin growth factor-1) treatment of neuronal cells from mutant mice rescued the soma size phenotype. We also found that mutation leads to down-regulation of the mTOR signaling pathway, known to be involved in neuronal size regulation. Our results suggest that i) reduced neuronal size is an important cellular phenotype of mutation in mice, and ii) MeCP2 might play a critical role in the maintenance of neuronal structure by modulation of the mTOR pathway. The definition of a quantifiable cellular phenotype supports using neuronal size as a biomarker in the development of a high-throughput, assay to screen for compounds that rescue small neuronal phenotype ("phenotypic assay").
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.12688/f1000research.8156.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5040159PMC
September 2016

Incontinentia pigmenti (Bloch-Sulzberger syndrome).

Handb Clin Neurol 2015 ;132:271-80

Neurology Research, Barrow Neurological Institute, Phoenix, AZ, USA; Center for Rare Childhood Disorders, Translational Genomics Research Institute, Phoenix, AZ, USA; Arizona Pediatric Neurology and Neurogenetics Associates, Phoenix, AZ, USA. Electronic address:

Incontinentia pigmenti (IP; Bloch-Sulzberger syndrome; OMIM #308300) is an X-linked dominant neurocutaneous disorder with presumed male lethality. It is usually diagnosed in female newborns based on skin features (erythematous, vesicular, or bullous eruption in linear streaks). The skin lesions evolve into a verrucous stage, followed by atrophy and scarring, leaving linear areas of hypopigmentation and hyperpigmented macules in bizarre patterns following Blaschko's lines. Systemic and neurologic complications include focal seizures and hemorrhagic cerebral infarction in infants, and retinal vasculopathy leading to blindness. Hypodontia, conical or pegged teeth, and linear areas of alopecia persist into adulthood. IP is caused by mutation of the IKBKG/NEMO gene on Xq28. Deletion of exons 4 to 10 (NEMOΔ4-10) accounts for about 80% of cases (familial and sporadic). NEMO mutation leads to loss of function of NF-κB, a critical protein that modulates cellular proliferation, apoptosis, and response to proinflammatory factors, leading to the characteristic features of IP. In female carriers, selective loss of cells expressing the mutant X-chromosome results in completely skewed X-inactivation in the majority of cases. Study of mouse models in which various components of the NF-κB pathway (including NEMO) have been knocked out has contributed significantly to our understanding of disease pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/B978-0-444-62702-5.00020-2DOI Listing
August 2016

A De Novo Mutation in TEAD1 Causes Non-X-Linked Aicardi Syndrome.

Invest Ophthalmol Vis Sci 2015 Jun;56(6):3896-904

Dorrance Center for Rare Childhood Disorders, Translational Genomics Research Institute, Phoenix, Arizona, United States 2Neurogenomics Division, Translational Genomics Research Institute, Phoenix, Arizona, United States.

Purpose: Aicardi syndrome (AIC) is a congenital neurodevelopmental disorder characterized by infantile spasms, agenesis of the corpus callosum, and chorioretinal lacunae. Variation in phenotype and disease severity is well documented, but chorioretinal lacunae represent the most constant pathological feature. Aicardi syndrome is believed to be an X-linked-dominant disorder occurring almost exclusively in females, although 46, XY males with AIC have been described. The purpose of this study is to identify genetic factors and pathways involved in AIC.

Methods: We performed exome/genome sequencing of 10 children diagnosed with AIC and their parents and performed RNA sequencing on blood samples from nine cases, their parents, and unrelated controls.

Results: We identified a de novo mutation in autosomal gene TEAD1, expressed in the retina and brain, in a patient with AIC. Mutations in TEAD1 have previously been associated with Sveinsson's chorioretinal atrophy, characterized by chorioretinal degeneration. This demonstrates that TEAD1 mutations can lead to different chorioretinal complications. In addition, we found that altered expression of genes associated with synaptic plasticity, neuronal development, retinal development, and cell cycle control/apoptosis is an important underlying potential pathogenic mechanism shared among cases. Last, we found a case with skewed X inactivation, supporting the idea that nonrandom X inactivation might be important in AIC.

Conclusions: We expand the phenotype of TEAD1 mutations, demonstrate its importance in chorioretinal complications, and propose the first putative pathogenic mechanisms underlying AIC. Our data suggest that AIC is a genetically heterogeneous disease and is not restricted to the X chromosome, and that TEAD1 mutations may be present in male patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/iovs.14-16261DOI Listing
June 2015

Diabetic Macular Edema: Pathophysiology and Novel Therapeutic Targets.

Ophthalmology 2015 Jul 30;122(7):1375-94. Epub 2015 Apr 30.

T-Gen Institute, Phoenix, Arizona.

Diabetic macular edema (DME) is the major cause of vision loss in diabetic persons. Alteration of the blood-retinal barrier is the hallmark of this disease, characterized by pericyte loss and endothelial cell-cell junction breakdown. Recent animal and clinical studies strongly indicate that DME is an inflammatory disease. Multiple cytokines and chemokines are involved in the pathogenesis of DME, with multiple cellular involvement affecting the neurovascular unit. With the introduction of anti-vascular endothelial growth factor (VEGF) agents, the treatment of DME has been revolutionized, and the indication for laser therapy has been limited. However, the response to anti-VEGF drugs in DME is not as robust as in proliferative diabetic retinopathy, and many patients with DME do not show complete resolution of fluid despite multiple intravitreal injections. Potential novel therapies targeting molecules other than VEGF and using new drug-delivery systems currently are being developed and evaluated in clinical trials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ophtha.2015.03.024DOI Listing
July 2015

Chemokine mediated monocyte trafficking into the retina: role of inflammation in alteration of the blood-retinal barrier in diabetic retinopathy.

PLoS One 2014 20;9(10):e108508. Epub 2014 Oct 20.

Department of Surgery, University of New Mexico School of Medicine, Albuquerque, New Mexico, United States of America; New Mexico VA Health Care System, Albuquerque, New Mexico, United States of America.

Inflammation in the diabetic retina is mediated by leukocyte adhesion to the retinal vasculature and alteration of the blood-retinal barrier (BRB). We investigated the role of chemokines in the alteration of the BRB in diabetes. Animals were made diabetic by streptozotocin injection and analyzed for gene expression and monocyte/macrophage infiltration. The expression of CCL2 (chemokine ligand 2) was significantly up-regulated in the retinas of rats with 4 and 8 weeks of diabetes and also in human retinal endothelial cells treated with high glucose and glucose flux. Additionally, diabetes or intraocular injection of recombinant CCL2 resulted in increased expression of the macrophage marker, F4/80. Cell culture impedance sensing studies showed that purified CCL2 was unable to alter the integrity of the human retinal endothelial cell barrier, whereas monocyte conditioned medium resulted in significant reduction in cell resistance, suggesting the relevance of CCL2 in early immune cell recruitment for subsequent barrier alterations. Further, using Cx3cr1-GFP mice, we found that intraocular injection of CCL2 increased retinal GFP+ monocyte/macrophage infiltration. When these mice were made diabetic, increased infiltration of monocytes/macrophages was also present in retinal tissues. Diabetes and CCL2 injection also induced activation of retinal microglia in these animals. Quantification by flow cytometry demonstrated a two-fold increase of CX3CR1+/CD11b+ (monocyte/macrophage and microglia) cells in retinas of wildtype diabetic animals in comparison to control non-diabetic ones. Using CCL2 knockout (Ccl2-/-) mice, we show a significant reduction in retinal vascular leakage and monocyte infiltration following induction of diabetes indicating the importance of this chemokine in alteration of the BRB. Thus, CCL2 may be an important therapeutic target for the treatment of diabetic macular edema.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0108508PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4203688PMC
July 2015

Epigenetics, autism spectrum, and neurodevelopmental disorders.

Neurotherapeutics 2013 Oct;10(4):742-56

Developmental Neurogenetics Laboratory, Barrow Neurological Institute, Phoenix, AZ, 85013, USA.

Epigenetic marks are modifications of DNA and histones. They are considered to be permanent within a single cell during development, and are heritable across cell division. Programming of neurons through epigenetic mechanisms is believed to be critical in neural development. Disruption or alteration in this process causes an array of neurodevelopmental disorders, including autism spectrum disorders (ASDs). Recent studies have provided evidence for an altered epigenetic landscape in ASDs and demonstrated the central role of epigenetic mechanisms in their pathogenesis. Many of the genes linked to the ASDs encode proteins that are involved in transcriptional regulation and chromatin remodeling. In this review we highlight selected neurodevelopmental disorders in which epigenetic dysregulation plays an important role. These include Rett syndrome, fragile X syndrome, Prader-Willi syndrome, Angelman syndrome, and Kabuki syndrome. For each of these disorders, we discuss how advances in our understanding of epigenetic mechanisms may lead to novel therapeutic approaches.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s13311-013-0227-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3805864PMC
October 2013

The role of monocyte subsets in myocutaneous revascularization.

J Surg Res 2013 Aug 6;183(2):963-75. Epub 2013 Mar 6.

Department of Surgery, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA.

Background: The controlled recruitment of monocytes from the circulation to the site of injury and their differentiation into tissue macrophages are critical events in the reconstitution of tissue integrity. Subsets of monocytes/macrophages have been implicated in the pathogenesis of atherosclerosis and tumor vascularity; however, the significance of monocyte heterogeneity in physiologic neovascularization is just emerging.

Materials And Methods: A cranial-based, peninsular-shaped myocutaneous flap was surgically created on the dorsum of wild-type mice (C57BL6) and populations of mice with genetic deletion of subset-specific chemokine ligand-receptor axes important in monocyte trafficking and function (CCL2(-/-) and CX3CR1(-/-)) (n=36 total; 12 mice per group, nine with flap and three unoperated controls). Planimetric analysis of digital photographic images was utilized to determine flap surface viability in wild-type and knockout mice. Real-time myocutaneous flap perfusion and functional revascularization was determined by laser speckle contrast imaging. Image analysis of CD-31 immunostained sections confirmed flap microvascular density and anatomy. Macrophage quantification and localization in flap tissues was determined by F4/80 gene and protein expression. Quantitative reverse transcription-polymerase chain reaction was performed on nonoperative back skin and postoperative flap tissue specimens to determine local gene expression.

Results: Myocutaneous flaps created on wild type and CX3CR1(-/-) mice were engrafted to the recipient site, resulting in viability. In contrast, distal full thickness cutaneous necrosis and resultant flap dehiscence was evident by d 10 in CCL2(-/-) mice. Over 10 d, laser speckle contrast imaging documented immediate graded flap ischemia in all three groups of mice, functional flap revascularization in wild type and CX3CR1(-/-) mice, and lack of distal flap reperfusion in CCL2(-/-) mice. Immunostaining of serial histologic specimens confirmed marked increases in microvascular density and number of macrophages in wild type mice, intermediate increases in CX3CR1(-/-) mice, and no significant change in vessel count or macrophage quantity in CCL2(-/-) mice over the study interval. Finally, quantitative reverse transcriptase polymerase chain reaction demonstrated that the loss of function of chemokine ligand and receptor genes influenced the transcription of local genes involved in monocyte chemotaxis and wound angiogenesis.

Conclusions: In a graded-ischemia wound healing model, monocyte recruitment was severely impaired in CCL2(-/-) mice, resulting in failure of flap revascularization and concomitant cutaneous necrosis. Analysis of CX3CR1-deficient mice revealed adequate monocyte recruitment and revascularization for flap survival; however, the myeloid cell response and magnitude of neovascularization were dampened compared with wild type mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jss.2013.02.019DOI Listing
August 2013

Diabetic retinopathy and inflammation: novel therapeutic targets.

Middle East Afr J Ophthalmol 2012 Jan;19(1):52-9

Department of Cell Biology and Physiology, University of New Mexico School of Medicine, Albuquerque, NM 87131.

Most anti-vascular endothelial growth factor (VEGF) therapies in diabetic macular edema are not as robust as in proliferative diabetic retinopathy. Although the VEGF appears to be a good target in diabetic macular edema, the anti-VEGF therapies appear to be of transient benefit as the edema recurs within a few weeks, and repeated injections are necessary. There is new evidence that indicates 'retinal inflammation' as an important player in the pathogenesis of diabetic retinopathy. There are common sets of inflammatory cytokines that are upregulated in both the serum and vitreous and aqueous samples, in subjects with diabetic retinopathy, and these cytokines can have multiple interactions to impact the pathogenesis of the disease. The key inflammatory events involved in the blood retinal barrier (BRB) alteration appear to be: (1) Increased expression of endothelial adhesion molecules such as ICAM1, VCAM1, PECAM-1, and P-selectin, (2) adhesion of leukocytes to the endothelium, (3) release of inflammatory chemokines, cytokines, and vascular permeability factors, (4) alteration of adherens and tight junctional proteins between the endothelial cells, and (5) infiltration of leukocytes into the neuro-retina, resulting in the alteration of the blood retinal barrier (diapedesis). VEGF inhibition itself may not achieve neutralization of other inflammatory molecules involved in the inflammatory cascade of the breakdown of the BRB. It is possible that the novel selective inhibitors of the inflammatory cascade (like angiopoietin-2, TNFα, and chemokines) may be useful therapeutic agents in the treatment of diabetic macular edema (DME), either alone or in combination with the anti-VEGF drugs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4103/0974-9233.92116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277025PMC
January 2012

Pericyte-derived sphingosine 1-phosphate induces the expression of adhesion proteins and modulates the retinal endothelial cell barrier.

Arterioscler Thromb Vasc Biol 2011 Dec 22;31(12):e107-15. Epub 2011 Sep 22.

Department of Cell Biology and Physiology, University of New Mexico School of Medicine, Albuquerque, New Mexico, USA.

Objective: The mechanisms that regulate the physical interaction of pericytes and endothelial cells and the effects of these interactions on interendothelial cell junctions are not well understood. We determined the extent to which vascular pericytes could regulate pericyte-endothelial adhesion and the consequences that this disruption might have on the function of the endothelial barrier.

Methods And Results: Human retinal microvascular endothelial cells were cocultured with pericytes, and the effect on the monolayer resistance of endothelial cells and expression of the cell junction molecules N-cadherin and VE-cadherin were measured. The molecules responsible for the effect of pericytes or pericyte-conditioned media on the endothelial resistance and cell junction molecules were further analyzed. Our results indicate that pericytes increase the barrier properties of endothelial cell monolayers. This barrier function is maintained through the secretion of pericyte-derived sphingosine 1-phosphate. Sphingosine 1-phosphate aids in maintenance of microvascular stability by upregulating the expression of N-cadherin and VE-cadherin, and downregulating the expression of angiopoietin 2.

Conclusions: Under normal circumstances, the retinal vascular pericytes maintain pericyte-endothelial contacts and vascular barrier function through the secretion of sphingosine 1-phosphate. Alteration of pericyte-derived sphingosine 1-phosphate production may be an important mechanism in the development of diseases characterized by vascular dysfunction and increased permeability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/ATVBAHA.111.235408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3225006PMC
December 2011

A potential role for angiopoietin 2 in the regulation of the blood-retinal barrier in diabetic retinopathy.

Invest Ophthalmol Vis Sci 2011 Jun 1;52(6):3784-91. Epub 2011 Jun 1.

Department of Cell Biology and Physiology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131, USA.

Purpose: Although VEGF has been identified as an important mediator of the blood-retinal barrier alteration in diabetic retinopathy, the hypothesis for this study was that that other molecules, including the angiopoietins (Ang-1 and -2), may play a role. The expression of angiopoietins was analyzed in an animal model of diabetic retinopathy, and the role of Ang-2 in the regulation of diabetes-induced alterations of vascular permeability was characterized.

Methods: Diabetes was induced in rats, and human retinal endothelial cells (HRECs) were grown in media with 5.5 or 30.5 mM glucose. Levels of Ang-1 and -2 mRNA and protein were analyzed. Fluorescence-based assays were used to assess the effect of Ang-2 on vascular permeability in vivo and in vitro. The effect of Ang-2 on VE-cadherin function was assessed by measuring the extent of tyrosine phosphorylation.

Results: Ang-2 mRNA and protein increased in the retinal tissues after 8 weeks of diabetes and in high-glucose-treated cells. Intravitreal injection of Ang-2 in rats produced a significant increase in retinal vascular permeability. Ang-2 increased HREC monolayer permeability that was associated with a decrease in VE-cadherin and a change in monolayer morphology. High glucose and Ang-2 produced a significant increase in VE-cadherin phosphorylation. CONCLUSIONS; Ang-2 is upregulated in the retina in an animal model of diabetes, and hyperglycemia induces the expression of Ang-2 in isolated retinal endothelial cells. Increased Ang-2 alters VE-cadherin function, leading to increased vascular permeability. Thus, Ang-2 may play an important role in increased vasopermeability in diabetic retinopathy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/iovs.10-6386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3109054PMC
June 2011

Oxidative stress is independently associated with non-alcoholic fatty liver disease (NAFLD) in subjects with and without type 2 diabetes.

Clin Biochem 2010 Jul 14;43(10-11):815-21. Epub 2010 Apr 14.

Madras Diabetes Research Foundation, Gopalapuram, Chennai, India.

Objective: Our work is aimed at exploring the interrelationship of oxidative stress and insulin resistance in NAFLD subjects with and without type 2 diabetes in a population-based study.

Methods: Subjects [n=200] were recruited from the Chennai Urban Rural Epidemiology Study. 1: Normal glucose tolerance (NGT) subjects without NAFLD; 2: NGT with NAFLD; 3: type 2 diabetic subjects [T2DM] without NAFLD and 4: T2DM with NAFLD. Thiobarbituric acid reactive substances (TBARS), protein carbonyl (PCC) and glutathione levels were measured by standard methods. Ultrasound of the liver was used to diagnose NAFLD.

Results: TBARS and PCC levels were significantly elevated and GSH/GSSG ratio was significantly decreased in diabetic subjects with NAFLD compared to all other groups (p trend <0.001). Oxidative stress markers significantly associated with NAFLD even after adjusting for age, gender, BMI and glycemic status.

Conclusions: Increased oxidative stress is independently associated with NAFLD in Asian Indians without and with T2DM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.clinbiochem.2010.04.003DOI Listing
July 2010

Association of leukocyte count with varying degrees of glucose intolerance in Asian Indians: the Chennai Urban Rural Epidemiology Study (CURES-26).

Metab Syndr Relat Disord 2009 Jun;7(3):205-10

Dr. Mohan's Diabetes Specialities Centre and Madras Diabetes Research Foundation, WHO Collaborating Centre for Noncommunicable Disease Prevention and Control, Gopalapuram, Chennai, India.

Objective: This study assessed the association of leukocyte count with different grades of glucose intolerance in Asian Indian subjects.

Methods: Three groups of subjects were recruited from the Chennai Urban Rural Epidemiology Study (CURES), a population-based study, representative of Chennai (formerly Madras), a city in southern India. Group 1 represented normal glucose tolerance (NGT) (n = 840), group 2 included impaired glucose tolerance (IGT) (n = 180), and group 3 included type 2 diabetes (n = 1170). Anthropometric measurements including weight, height, and waist measurements were obtained using standardized techniques. Leukocyte count was measured by an automated flow cytometry instrument (Sysmex SF-3000, Japan). Fasting insulin was measured by enzyme-linked immunosorbent assay and insulin resistance was calculated using the homeostasis model assessment (HOMA-IR).

Results: Subjects with diabetes (8.0 +/- 1.5 x 10(3)/microL) and IGT (7.9 +/- 1.3 x 10(3)/microL) had a significantly higher mean leukocyte count compared to the NGT group (7.4 +/- 1.5 x 10(3)/microL) (P < 0.001). Leukocyte count was significantly increased in NGT subjects with insulin resistance (IR) as measured by HOMA-IR (7.5 +/- 1.5 x 10(3)/microL; P < 0.001) compared to NGT subjects without IR (7.0 +/- 1.4 x 10(3)/microL). Regression analysis showed that there was a linear increase in mean leukocyte count with increasing severity of glucose intolerance, even after adjusting for age, waist circumference, and HOMA-IR.

Conclusions: Among Asian Indians who are known to have high risk of premature coronary artery disease and diabetes, a significant association exists between leukocyte count and glucose intolerance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/met.2008.0024DOI Listing
June 2009

A common nonsynonymous single nucleotide polymorphism in the SLC30A8 gene determines ZnT8 autoantibody specificity in type 1 diabetes.

Diabetes 2008 Oct 30;57(10):2693-7. Epub 2008 Jun 30.

Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, Aurora, Colorado, USA.

Objective: Zinc transporter eight (SLC30A8) is a major target of autoimmunity in human type 1A diabetes and is implicated in type 2 diabetes in genome-wide association studies. The type 2 diabetes nonsynonymous single nucleotide polymorphism (SNP) affecting aa(325) lies within the region of highest ZnT8 autoantibody (ZnT8A) binding, prompting an investigation of its relationship to type 1 diabetes.

Research Design And Methods: ZnT8A radioimmunoprecipitation assays were performed in 421 new-onset type 1 diabetic Caucasians using COOH-terminal constructs incorporating the known human aa(325) variants (Trp, Arg, and Gln). Genotypes were determined by PCR-based SNP analysis. RESULTS-Sera from 224 subjects (53%) were reactive to Arg(325) probes, from 185 (44%) to Trp(325)probes, and from 142 (34%) to Gln(325)probes. Sixty subjects reacted only with Arg(325) constructs, 31 with Trp(325) only, and 1 with Gln(325) only. The restriction to either Arg(325) or Trp(325) corresponded with inheritance of the respective C- or T-alleles. A strong gene dosage effect was also evident because both Arg- and Trp-restricted ZnT8As were less prevalent in heterozygous than homozygous individuals. The SLC30A8 SNP allele frequency (75% C and 25% T) varied little with age of type 1 diabetes onset or the presence of other autoantibodies.

Conclusions: The finding that diabetes autoimmunity can be defined by a single polymorphic residue has not previously been documented. It argues against ZnT8 autoimmunity arising from molecular mimicry and suggests a mechanistic link between the two major forms of diabetes. It has implications for antigen-based therapeutic interventions because the response to ZnT8 administration could be protective or immunogenic depending on an individual's genotype.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2337/db08-0522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2551679PMC
October 2008

Advanced glycation index and its association with severity of diabetic retinopathy in type 2 diabetic subjects.

J Diabetes Complications 2008 Jul-Aug;22(4):261-6. Epub 2008 Apr 16.

Madras Diabetes Research Foundation and Dr. Mohan's Diabetes Specialities Center, Gopalapuram, Chennai, India.

Background: This study investigates the association of advanced glycation index (AGI), a simple assay to detect advanced glycation endproducts (AGEs) in serum, with severity of diabetic retinopathy (DR) in type 2 diabetic subjects.

Methods: The study included 188 type 2 diabetic subjects without DR, 153 subjects with nonproliferative DR, 41 subjects with proliferative DR, and 188 control participants. Serum levels of AGEs were monitored with a spectrofluorimeter by recording Maillard-specific fluorescence.

Results: AGI values increased with severity of DR (analysis of variance, P<.0001). Among diabetic subjects, AGI (mean+/-S.E.) was higher among subjects with nonproliferative diabetic retinopathy (NPDR; 6.7+/-0.1 U) and proliferative diabetic retinopathy (PDR; 9.1+/-0.3 U) than among subjects without DR (P<.0001). By arranging the levels of serum AGI in quartiles, the proportion of PDR subjects increased with increasing AGI values, with maximum subjects in the last quartile (trend chi(2)=60.239, P<.0001). AGI was associated with NPDR even after adjusting for age, gender, duration of diabetes, and glycated hemoglobin [odds ratio (OR)=1.33; 95% confidence interval (95% CI)=1.12-1.57; P=.001]. Similarly, AGI showed a significant association with PDR even after adjusting for various risk factors (OR=2.47; 95% CI=1.75-3.47; P<.0001). Receiver-operating-characteristics curve analysis revealed that the threshold level of 8.07 U had a 78% sensitivity, an 83.6% specificity, and an 86.1% accuracy for detecting PDR.

Conclusion: AGI showed a significant association with the severity of DR and, hence, could be used as a prognostic tool to predict the development and progression of DR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jdiacomp.2007.05.005DOI Listing
August 2008

Glutamine fructose-6-phosphate amidotransferase (GFAT) gene expression and activity in patients with type 2 diabetes: inter-relationships with hyperglycaemia and oxidative stress.

Clin Biochem 2007 Sep 24;40(13-14):952-7. Epub 2007 May 24.

Madras Diabetes Research Foundation and Dr. Mohan's Diabetes Specialties Center 6B, Conran Smith Road, Gopalapuram, Chennai, India.

Objective: : Cell culture and animal model studies have strongly suggested a role for the rate-limiting enzyme for hexosamine biosynthesis, glutamine:fructose-6-phosphate amidotransferase (GFAT) in insulin resistance. However, there are very few clinical studies and none on Asian Indians, a high-risk group for type 2 diabetes (T2DM), which examined the role of GFAT in insulin resistance and T2DM.

Design And Method: : The study group comprised of T2DM subjects without any complications (n=25) and control non-diabetic subjects (n=23). GFAT mRNA expression and activity were measured by semi-quantitative RT-PCR and fluorimetry, respectively. Oxidative damage was assessed in plasma by the extent of lipid peroxidation [thiobarbituric acid reactive substances (TBARS)] and protein carbonyl content (PCO) using standard methods.

Result: : The mean (+/-SE) GFAT activity was significantly higher in diabetic (30.22+/-2.40 pM/mg protein/min) compared to control subjects (20.10+/-1.12 pM/mg protein/min) (p<0.001). Plasma levels of diabetic patients also exhibited increased lipid peroxidation and protein carbonylation. GFAT activity was positively correlated (p<0.005) with GFAT mRNA, HbA(1c), insulin resistance (HOMA-IR), postprandial plasma glucose and levels of TBARS and PCO. In multiple logistic regression analysis, the association between GFAT activity and T2DM persisted even after adjusting for age, gender, BMI and HOMA-IR (OR=1.202, p=0.026).

Conclusion: : Increased GFAT activity appears to be associated with insulin resistance, postprandial hyperglycaemia and oxidative stress in T2DM and may point towards a potential pathway amenable for therapeutic intervention.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.clinbiochem.2007.05.002DOI Listing
September 2007

Is insulin signaling molecules misguided in diabetes for ubiquitin-proteasome mediated degradation?

Mol Cell Biochem 2005 Jul;275(1-2):117-25

Department of Cell and Molecular Biology, Madras Diabetes Research Foundation, 6B, Conran Smith Road, Gopalapuram, Chennai 600 086, India.

Recent mining of the human and mouse genomes, use of yeast genetics, and detailed analyses of several biochemical pathways, have resulted in the identification of many new roles for ubiquitin-proteasome mediated degradation of proteins. In the context of last year's award of Noble Prize (Chemistry) work, the ubiquitin and ubiquitin-like modifications are increasingly recognized as key regulatory events in health and disease. Although the ATP-dependent ubiquitin-proteasome system has evolved as premier cellular proteolytic machinery, dysregulation of this system by several different mechanisms leads to inappropriate degradation of specific proteins and pathological consequences. While aberrations in the ubiquitin-proteasome pathway have been implicated in certain malignancies and neurodegenerative disorders, recent studies indicate a role for this system in the pathogenesis of diabetes and its complications. Inappropriate degradation of insulin signaling molecules such as insulin receptor substrates (IRS-1 and IRS-2) has been demonstrated in experimental diabetes, mediated in part through the up-regulation of suppressors of cytokine signaling (SOCS). It appears that altered ubiquitin-proteasome system might be one of the molecular mechanisms of insulin resistance in many pathological situations. Drugs that modulate the SOCS action and/or proteasomal degradation of proteins could become novel agents for the treatment of insulin resistance and Type 2 diabetes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11010-005-1083-yDOI Listing
July 2005
-->