Understanding the Molecular Basis of Fragile X Syndrome Using Differentiated Mesenchymal Stem Cells

Abstract Objectives Fragile X syndrome (FXS) has been known as the most common cause of inherited intellectual disability and autism. This disease results from the loss of fragile X mental retardation protein expression due to the expansion of CGG repeats located on the 5’ untranslated region of the fragile X mental retardation 1 (FMR1) gene. Materials & Methods In the present study, the peripheral blood-mesenchymal stem cells (PB-MSCs) of two female full mutation carriers were differentiated into neuronal cells by the suppression of bone morphogenesis pathwaysignaling. Then, the expression of genes adjacent to CGG repeats expansion, including SLIT and NTRK-like protein 2 (SLITRK2), SLIT and NTRK-like protein 4 (SLITRK4), methyl CpG binding protein 2 (MECP2), and gamma-aminobutyric acid receptor subunit alpha-3 (GABRA3), were evaluated in these cells using SYBR Green real-time polymerase chain reaction. Results The obtained results indicated that the expression of SLITRK2 and SLITRK4 were upregulated and downregulated in the neuron-like cells differentiated from the PB-MSCs of females with FMR1 full mutation, compared to that of the normal females, respectively. Furthermore, the expression of MECP2 and GABRA3 genes were observed to be related to the phenotypic differences observed in the female FMR1full mutation carriers Conclusion The observed association of expression of genes located upstream of the FMR1 gene with phenotypic differences in the female carriers could increase the understanding of novel therapeutic targets for patients with mild symptoms of FXS and the patients affected by other FMR1-related disorder

The liver-derived exosomes stimulate insulin gene expression in pancreatic beta cells under condition of insulin resistance

Introduction: An insufficient functional beta cell mass is a core pathological hallmark of type 2 diabetes (T2D). Despite the availability of several effective pharmaceuticals for diabetes management, there is an urgent need for novel medications to protect pancreatic beta cells under diabetic conditions. Integrative organ cross-communication controls the energy balance and glucose homeostasis. The liver and pancreatic islets have dynamic cross-communications where the liver can trigger a compensatory beta cell mass expansion and enhanced hormonal secretion in insulin-resistant conditions. However, the indispensable element(s) that foster beta cell proliferation and insulin secretion have yet to be completely identified. Exosomes are important extracellular vehicles (EVs) released by most cell types that transfer biological signal(s), including metabolic messengers such as miRNA and peptides, between cells and organs. Methods: We investigated whether beta cells can take up liver-derived exosomes and examined their impact on beta cell functional genes and insulin expression. Exosomes isolated from human liver HepG2 cells were characterized using various methods, including Transmission Electron Microscopy (TEM), dynamic light scattering (DLS), and Western blot analysis of exosomal markers. Exosome labeling and cell uptake were assessed using CM-Dil dye. The effect of liver cell-derived exosomes on Min6 beta cells was determined through gene expression analyses of beta cell markers and insulin using qPCR, as well as Akt signaling using Western blotting. Results: Treatment of Min6 beta cells with exosomes isolated from human liver HepG2 cells treated with insulin receptor antagonist S961 significantly increased the expression of beta cell markers Pdx1, NeuroD1, and Ins1 compared to the exosomes isolated from untreated cells. In line with this, the activity of AKT kinase, an integral component of the insulin receptor pathway, is elevated in pancreatic beta cells, as represented by an increase in AKT’s downstream substrate, FoxO1 phosphorylation. Discussions: This study suggests that liver-derived exosomes may carry a specific molecular cargo that can affect insulin expression in pancreatic beta cells, ultimately affecting glucose homeostasis

A novel deletion mutation in ASPM gene in an Iranian family with autosomal recessive primary microcephaly

How to Cite This Article: Akbarizar E, Ebrahimpour M, Akbari S, Arzhanghi S, Abedini SS, Najmabadi H, Kahrizi K. A Novel Deletion Mutation in ASPM Gene in an Iranian Family with Autosomal Recessive Primary Microcephaly. Iran J Child Neurol.  2013 Spring;7(2):23-30. ObjectiveAutosomal recessive primary microcephaly (MCPH) is a neurodevelopmental and genetically heterogeneous disorder with decreased head circumference due to the abnormality in fetal brain growth. To date, nine loci and nine genes responsible for the situation have been identified. Mutations in the ASPM gene (MCPH5) is the most common cause of MCPH. The ASPM gene with 28 exons is essential for normal mitotic spindle function in embryonic neuroblasts.Materials & MethodsWe have ascertained twenty-two consanguineous families withintellectual disability and different ethnic backgrounds from Iran. Ten out of twenty-two families showed primary microcephaly in clinical examination. We investigated MCPH5 locus using homozygosity mapping by microsatellite marker. ResultSequence analysis of exon 8 revealed a deletion of nucleotide (T) in donor site of splicing site of ASPM in one family. The remaining nine families were not linked to any of the known loci. More investigation will be needed to detect the causative defect in these families.ConlusionWe detected a novel mutation in the donor splicing site of exon 8 of the ASPM gene. This deletion mutation can alter the ASPM transcript leading to functional impairment of the gene product. References1. Pattison L, Crow YJ, Deeble VJ, Jackson AP, Jafri H, Rashid Y, et al. A Fifth Locus for Primary Autosomal Recessive Microcephaly Maps to Chromosome 1q31. Am J Hum Genet 2000;67(6):1578-80.2. Darvish H, Esmaeeli-Nieh S, Monajemi G, Mohseni M, Ghasemi-Firouzabadi S, Abedini S, et al. A clinical and molecular genetic study of 112 Iranian families with primary microcephaly. Journal of Medical Genetics 2010;47(12):823-8.3. Tolmie JL, M M, JB S, D D, JM C. Microcephaly: genetic counselling and antenatal diagnosis after the birth of an affected child. Am JMed Genet 1987;27583-94.4. Cowie V. The genetics and sub-classification of microcephaly. J Ment Defic Res 1960;4:42-7. 5. Woods C. Human microcephaly. Curr Opin Neurobiol 2004;14(1):112-7.6. Kaindl AM PS, Kumar P, Kraemer N, Issa L, Zwirner A, Gerard B, Verloes A MS,et al.Many roads lead to primary autosomal recessive microcephaly. Prog Neurobiol 2010;90:363-83.7. Kumar A BS, Babu M, Markandaya M, Girimaji SC. Genetic analysis of primary microcephaly in Indian families: novel ASPM mutations. Clin Genet 2004;66:341-8.8. Jackson AP, Eastwood H, Bell SM, Adu J, Toomes C, Carr IM, et al. Identification of microcephalin, a protein implicated in determining the size of the human brain. The American Journal of Human Genetics 2002;71(1):136-42.9. Roberts E, Jackson AP, Carradice AC, Deeble VJ, Mannan J, Rashid Y, et al. The second locus for autosomal recessive primary microcephaly (MCPH2) maps to chromosome 19q13. 1-13.2. European journal of human genetics: EJHG  1999;7(7):815.10. Kousar R, Hassan MJ, Khan B, Basit S, Mahmood S, Mir A, et al. Mutations in WDR62 gene in Pakistani families with autosomal recessive primary microcephaly. BMC neurology 2011;11(1):119.11. Evans PD, Vallender EJ, Lahn BT. Molecular evolutionof the brain size regulator genes<i> CDK5RAP2</i>and<i> CENPJ</i>. Gene 2006;375:75-9.12. Nagase T, Nakayama M, Nakajima D, Kikuno R, Ohara O. Prediction of the coding sequences of unidentified human genes. XX. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro. DNA research 2001;8(2):85-95. 13. Jamieson CR GC, Abramowicz MJ. Primary autosomal recessive microcephaly: homozygosity mapping of MCPH4 to chromosome 15. Am J Hum Genet 1999;65:1465-9.14. Genin A, Desir J, Lambert N, Biervliet M, Van Der Aa N, Pierquin G, et al. Kinetochore KMN network gene CASC5 mutated in Primary Microcephaly. Human molecular genetics 2012.15. Bond J, Roberts E, Mochida GH, Hampshire DJ, Scott S, Askham JM, et al. ASPM is a major determinant of cerebral cortical size. Nature genetics 2002;32(2):316-20.16. Fish JL, Kosodo Y, Enard W, Pääbo S, Huttner WB. Aspm specifically maintains symmetric proliferative divisions of neuroepithelial cells. Proceedings of the National Academy of Sciences 2006;103(27):10438-43.17. Leal G, Roberts E, Silva E, Costa S, Hampshire D, Woods C. A novel locus for autosomal recessive primary microcephaly (MCPH6) maps to 13q12.2. Journal of Medical Genetics 2003;40(7):540-2.18. Kumar A. Mutations in STIL, encoding a pericentriolar and centrosomal protein, cause primary microcephaly. The American Journal of Human Genetics 2009;84(2):286-90.19. Hussain MS, Baig SM, Neumann S, Nurnberg G, Farooq M, Ahmad I, et al. Atruncating mutation on CEP135 causes primary microcephaly and disturbed centrosomal function.AMJ,HumGenet 2012;90:871-8.20. Guernsey DL, Jiang H, Hussin J, Arnold M, Bouyakdan K, Perry S, et al. Mutations in centrosomal protein CEP152 in primary microcephaly families linked to MCPH4. The American Journal of Human Genetics 2010;87(1):40-51.21. Gul A, Hassan MJ, Mahmood S, Chen W, Rahmani S, Naseer MI, et al. Genetic studies of autosomal recessive primary microcephaly in 33 Pakistani families: novel sequence variants in ASPM gene. Neurogenetics 2006;7(2):105-10.22. Roberts E, Hampshire D, Springell K, Pattison L, Y C, Jafri H, et al. Autosomal recessive primary microcephaly: an analysis of locus heterogeneity and phenotypic variation. J Med Genet 2002;39:718–721.23. Woods CG BJ, Enard W. Autosomal recessive primary microcephaly (MCPH): a review of clinical, molecular, and evolutionary findings. Am J Hum Genet 2005 May;76(5):717-28.24. Kouprina N, Pavlicek A, Collins NK, Nakano M, Noskov VN, Ohzeki JI, et al. The microcephaly ASPM gene is expressed in proliferating tissues and encodes for a mitotic spindle protein. Human Molecular Genetics 2005;14(15):2155-65.25. Bond J, Scott S, Hampshire DJ, Springell K, Corry P, Abramowicz MJ, et al. Protein-Truncating Mutations in< i> ASPM</i> Cause Variable Reduction in Brain Size. The American Journal of Human Genetics 2003;73(5):1170-7.26. Pichon B, Vankerckhove S, Bourrouillou G, Duprez L, Abramowicz MJ. A translocation breakpoint disrupts the ASPM gene in a patient with primary microcephaly. European journal of Human Genetics 2004;12(5):419-21.27. Garshasbi.M, Motazacker M, Kahrizi K, Behjati F, Abedini S, Nieh S, et al. SNP array-based homozygosity mapping reveals MCPH1 deletion in family with autosomal recessive mental retardation and mild microcephaly. Hum Genet 2006 Feb;118(6):708-15.28. Jackson A, McHale D, Campbell D, Jafri H, Rashid Y, Mannan J, et al. Primary autosomal recessive microcephaly (MCPH1) maps to chromosome 8p22-pter. Am J Hum Genet 1998 Aug;63(2):541-6.29. Moynihan L, Jackson A, Roberts E, Karbani G, Lewis I, Corry P, et al. A third novel locus for primary autosomal recessive microcephaly maps to chromosome 9q34. Am J Hum Genet 2000 Feb;66(2):724-7.30. Bond J, Roberts E, Springell K, Lizarraga S, Scott S, Higgins J, et al. A centrosomalmechanism involving CDK5RAP2 and CENPJ controls brain size. Nat Genet.2005 Apr;37(4):353-5. Nat Genet 2005 Apr;37(4):353-5.31. Jamieson C, Govaerts C, Abramowicz M, J. Primary autosomal recessive microcephaly: homozygosity mapping of MCPH4 to chromosome 15. Am J Hum Genet. 1999;65:1465-9

Two novel SLC26A4 mutations in Iranian families with autosomal recessive hearing loss

Objective: Due to the fact that SLC26A4 has been suggested as the second cause of hearing loss (HL) in Iran as well as many other countries, obtaining more comprehensive information about SLC26A4 mutations can facilitate more efficient genetic services to the patients with hereditary hearing loss. This investigation aims to detect genetic cause of two Iranian families with hearing loss. Methods: In the present study, genetic linkage analysis via 4 short tandem repeat markers linked to SLC26A4 was performed for two consanguineous families originating from Hormozgan and Chaharmahal va Bakhtiari provinces of Iran, co-segregating autosomal recessive hearing loss and showed no GJB2 mutations in our preliminary investigation. For identification of mutations, DNA sequencing of SLC26A4 including all the 21 exons, exon-intron boundaries and the promoter was carried out. Results: The results showed linkage to this gene in both families. After sequencing, two novel SLC26A4 mutations (c.65-66insT in exon 2 and c.2106delG in exon 19) were revealed in the two studied families. Conclusion: Results of this study stress the necessity of considering the analysis of SLC26A4 in molecular diagnosis of deafness especially when phenotypes such as goiter or enlarged vestibular aqueduct are present. (C) 2012 Elsevier Ireland Ltd. All rights reserved

Comparative Efficacy of Bispectral Index Monitoring and Clinical Assessment in The Recovery of Patients Undergoing Open Renal Surgery: A Randomized, Double-Blind Study

Background and Aims: Maintaining the sufficient depth of anesthesia with an adequate anesthetic drug dosage in patients undergoingsurgery is one of the most significant issues. Inadequate depth of anesthesia can cause significant disturbances in hemodynamicparameters. In this study, clinical assessment and bispectral (BIS) index monitoring compare the depth of general anesthesiaand recovery time in patients undergoing open renal surgery.Method: In this double-blind, randomized, controlled trial, all patients undergoingopenrenal surgery were enrolledandrandomlydivided into a BIS group and clinical assessment group (control). In the BIS group, the electrodes of BIS monitoring system wereplaced on frontal and temporal lobes of the patient. The time of eye opening, verbal response to verbal stimulation, extubation time,the duration of stay in the recovery unit, the first-time of narcotic usage, and total dosage of intravenous narcotics were assessed in2 groups.Results: A total of 96 patients were enrolled. Sex, age, BMI, duration of surgery, length of stay in the recovery room and first-timenarcotic drug usage were not significantly different in the two groups. However, the length of time from the anesthetic drug discontinuationto eye opening, verbal responses to verbal stimulation and extubation was significantly lower in the BIS group thanthe control group, respectively (P = 0.002, P = 0.007, P = 0.019).Conclusions: The evaluation of the aneasthesia status of patients based on the BIS index would be more efficient in decreasing theemergence anaesthesia including eye opening, verbal response, extubation after anesthesi

ARHGEF9 disease: Phenotype clarification and genotype-phenotype correlation.

ObjectiveWe aimed to generate a review and description of the phenotypic and genotypic spectra of ARHGEF9 mutations.MethodsPatients with mutations or chromosomal disruptions affecting ARHGEF9 were identified through our clinics and review of the literature. Detailed medical history and examination findings were obtained via a standardized questionnaire, or if this was not possible by reviewing the published phenotypic features.ResultsA total of 18 patients (including 5 females) were identified. Six had de novo, 5 had maternally inherited mutations, and 7 had chromosomal disruptions. All females had strongly skewed X-inactivation in favor of the abnormal X-chromosome. Symptoms presented in early childhood with delayed motor development alone or in combination with seizures. Intellectual disability was severe in most and moderate in patients with milder mutations. Males with severe intellectual disability had severe, often intractable, epilepsy and exhibited a particular facial dysmorphism. Patients with mutations in exon 9 affecting the protein's PH domain did not develop epilepsy.ConclusionsARHGEF9 encodes a crucial neuronal synaptic protein; loss of function of which results in severe intellectual disability, epilepsy, and a particular facial dysmorphism. Loss of only the protein's PH domain function is associated with the absence of epilepsy

Hemoglobin Q-Iran detected in family members from Northern Iran: a case report

<p>Abstract</p> <p>Introduction</p> <p>Hemoglobin Q-Iran (α75Asp→His) is an important member of the hemoglobin Q family, molecularly characterized by the replacement of aspartic acid by histidine. The first report of hemoglobin Q-Iran and the nomenclature of this hemoglobinopathy dates back to 1970. Iran is known as a country with a high prevalence of α- and β-thalassemia and different types of hemoglobinopathy. Many of these variants are yet to be identified as the practice of molecular laboratory techniques is limited in this part of the world. Applying such molecular methods, we report the first hemoglobin Q-Iran cases in Northern Iran.</p> <p>Case presentation</p> <p>An unusual band was detected in an isoelectric focusing test and cellulose acetate electrophoresis of a sample from a 22-year-old Iranian man from Mazandaran Province. Capillary zone electrophoresis analysis identified this band as hemoglobin Q. A similar band was also detected in his mother's electrophoresis (38 years, Iranian ethnicity). The cases underwent molecular investigation and the presence of a hemoglobin Q-Iran mutation was confirmed by the amplification refractory mutation system polymerase chain reaction method. Direct conventional sequencing revealed a single guanine to cytosine missense mutation (c.226G > C; <it>G</it>AC ><it>C</it>AC) at codon 75 in the α-globin gene in both cases.</p> <p>Conclusion</p> <p>The wide spectrum and high frequency of nondeletional α-globin mutations in Mazandaran Province is remarkable and seem to differ considerably from what has been found in Mediterranean populations. This short communication reports the first cases of patients with hemoglobin Q found in that region.</p

Two Iranian families with a novel mutation in GJB2 causing autosomal dominant nonsyndromic hearing loss

Mutations in GJB2 , encoding connexin 26 (Cx26), cause both autosomal dominant and autosomal recessive nonsyndromic hearing loss (ARNSHL) at the DFNA3 and DFNB1 loci, respectively. Most of the over 100 described GJB2 mutations cause ARNSHL. Only a minority has been associated with autosomal dominant hearing loss. In this study, we present two families with autosomal dominant nonsyndromic hearing loss caused by a novel mutation in GJB2 (p.Asp46Asn). Both families were ascertained from the same village in northern Iran consistent with a founder effect. This finding implicates the D46N missense mutation in Cx26 as a common cause of deafness in this part of Iran mandating mutation screening of GJB2 for D46N in all persons with hearing loss who originate from this geographic region. © 2011 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/83755/1/33209_ftp.pd

Mutations in NSUN2 Cause Autosomal- Recessive Intellectual Disability

With a prevalence between 1 and 3%, hereditary forms of intellectual disability (ID) are among the most important problems in health care. Particularly, autosomal-recessive forms of the disorder have a very heterogeneous molecular basis, and genes with an increased number of disease-causing mutations are not common. Here, we report on three different mutations (two nonsense mutations, c.679C>T [p.Gln227∗] and c.1114C>T [p.Gln372∗], as well as one splicing mutation, g.6622224A>C [p.Ile179Argfs∗192]) that cause a loss of the tRNA-methyltransferase-encoding NSUN2 main transcript in homozygotes. We identified the mutations by sequencing exons and exon-intron boundaries within the genomic region where the linkage intervals of three independent consanguineous families of Iranian and Kurdish origin overlapped with the previously described MRT5 locus. In order to gain further evidence concerning the effect of a loss of NSUN2 on memory and learning, we constructed a Drosophila model by deleting the NSUN2 ortholog, CG6133, and investigated the mutants by using molecular and behavioral approaches. When the Drosophila melanogaster NSUN2 ortholog was deleted, severe short-term-memory (STM) deficits were observed; STM could be rescued by re-expression of the wild-type protein in the nervous system. The humans homozygous for NSUN2 mutations showed an overlapping phenotype consisting of moderate to severe ID and facial dysmorphism (which includes a long face, characteristic eyebrows, a long nose, and a small chin), suggesting that mutations in this gene might even induce a syndromic form of ID. Moreover, our observations from the Drosophila model point toward an evolutionarily conserved role of RNA methylation in normal cognitive development