ASSESSMENT OF GENETICS MUTATIONS IN SMPD1, NPC1, …
Transcript of ASSESSMENT OF GENETICS MUTATIONS IN SMPD1, NPC1, …
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ASSESSMENT OF GENETICS MUTATIONS IN SMPD1, NPC1, NPC2
GENES TO INDUCED NIEMANN–PICK SYNDROME
Shahin Asadi*1, Mahsa Jamali
2, Hamideh Mohammadzadeh
3 and Ali Nazirzadeh
4
1Molecular Genetics-IRAN-TABRIZ. Director of the Division of Medical Genetics and
Molecular Research.
2Master of Molecular Genetics.
3Master of Molecular Biology-Genetics.
4Ph. D of Biotechnology.
ABSTRACT
In this study we have analyzed 30 people. 10 patients Niemann–Pick
disease and 20 persons control group. The genes SMPD1, NPC1,
NPC2, analyzed in terms of genetic mutations made. In this study,
people who have genetic mutations were targeted, with nervous
disorders Niemann–Pick disease. In fact, of all people with Niemann–
Pick disease.. 10 patients Niemann–Pick disease had a genetic
mutations in the genes SMPD1, NPC1, NPC2 Niemann–Pick
disease.Any genetic mutations in the target genes control group, did
not show.
KEYWORDS: Genetic study, Niemann–Pick disease, Mutations the
gene SMPD1, NPC1, NPC2, RT-PCR.
INTRODUCATION
Niemann–Pick disease (/niːmənˈpɪk/nee-mən-PIK)[1]
is a group of inherited, severe
metabolic disorders in which sphingomyelin accumulates in lysosomes in cells. The
lysosomes normally transport material through and out of the cell.
The prognosis is individual, but the severe form is fatal in toddlerhood and, in some cases,
patients with the milder forms may have normal lifespans.
World Journal of Pharmaceutical Research SJIF Impact Factor 7.523
Volume 6, Issue 10, 1375-1394. Research Article ISSN 2277– 7105
Article Received on 24 July 2017,
Revised on 14 August 2017, Accepted on 03 Sept. 2017
DOI: 10.20959/wjpr201710-9432
8533
*Corresponding Author
Shahin Asadi
Molecular Genetics-IRAN-
TABRIZ. Director of the
Division of Medical
Genetics and Molecular
Research.
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This disease involves dysfunctional metabolism of sphingolipids, which are fats found in cell
membranes, so it is a kind of sphingolipidosis. Sphingolipidoses, in turn, are included in the
larger family of lysosomal storage diseases.[2]
Signs and Symptoms
Symptoms are related to the organs in which sphingomyelin accumulates. Enlargement of the
liver and spleen (hepatosplenomegaly) may cause reduced appetite, abdominal distension,
and pain. Enlargement of the spleen (splenomegaly) may also cause low levels of platelets in
the blood (thrombocytopenia).
Accumulation of sphingomyelin in the central nervous system (including the cerebellum)
results in unsteady gait (ataxia), slurring of speech (dysarthria), and difficulty in swallowing
(dysphagia). Basal ganglia dysfunction causes abnormal posturing of the limbs, trunk, and
face (dystonia). Upper brainstem disease results in impaired voluntary rapid eye movements
(supranuclear gaze palsy). More widespread disease involving the cerebral cortex and
subcortical structures causes gradual loss of intellectual abilities, causing dementia and
seizures.
Bones also may be affected: symptoms may include enlarged bone marrow cavities, thinned
cortical bone, or a distortion of the hip bone called coxa vara. Sleep-related disorders, such as
sleep inversion, sleepiness during the day and wakefulness at night, may occur. Gelastic
cataplexy, the sudden loss of muscle tone when the affected patient laughs, also is seen.
Niemann–Pick disease has an autosomal recessive pattern of inheritance.
Mutations in the SMPD1 gene cause Niemann–Pick disease types A and B. They produce a
deficiency in the activity of the lysosomal enzyme acid sphingomyelinase, that breaks down
the lipid sphingomyelin.[3]
Mutations in NPC1 or NPC2 cause Niemann–Pick disease, type C (NPC), which affects a
protein used to transport lipids.[3]
Type D originally was separated from type C to delineate a group of patients with otherwise
identical disorders who shared a common Nova Scotian ancestry. Patients in this group are
known to share a specific mutation in the NPC1 gene, so NPC is used for both groups. Before
the molecular defects were described, the terms "Niemann–Pick type I" and "Niemann–Pick
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type II" were proposed to separate the high- and low-sphingomyelin forms of the disease in
the early 1980s.
Niemann–Pick disease is inherited in an autosomal recessive pattern, which means both
copies, or both alleles, of the gene must be defective to cause the disease. "Defective" means
they are altered in a way that impairs their function. Most often, the parents of a child with an
autosomal recessive disorder are carriers: they have one copy of the altered gene, but are not
affected because the other copy produces the enzyme. If both parents are carriers, each
pregnancy has a 25% chance of producing an affected child. Genetic counseling and genetic
testing are recommended for families who may be carriers of Niemann–Pick.
Niemann–Pick diseases are a subgroup of lipid storage disorders called sphingolipidoses in
which harmful quantities of fatty substances, or lipids, accumulate in the spleen, liver, lungs,
bone marrow, and brain.
In the classic infantile type-A variant, a missense mutation causes complete deficiency of
sphingomyelinase. Sphingomyelin is a component of cell membrane including the organellar
membrane, so the enzyme deficiency blocks degradation of lipid, resulting in the
accumulation of sphingomyelin within lysosomes in the macrophage-monocyte phagocyte
lineage. Affected cells become enlarged, sometimes up to 90 μm in diameter, secondary to
the distention of lysosomes with sphingomyelin and cholesterol. Histology shows lipid-laden
macrophages in the marrow and "sea-blue histiocytes" on pathology. Numerous small
vacuoles of relatively uniform size are created, giving the cytoplasm a foamy appearance.
PATHOLOGY
Loss of myelin in the central nervous system is considered to be a main pathogenic factor.
Research uses animal models carrying the underlying mutation for Niemann–Pick disease,
e.g. a mutation in the NPC1 gene as seen in Niemann-Pick type C disease. In this model, the
expression of myelin gene regulatory factor (MRF) has been shown to be significantly
decreased.[15]
MRF is a transcription factor of critical importance in the development and
maintenance of myelin sheaths.[16]
A perturbation of oligodendrocyte maturation and the
myelination process might, therefore, be an underlying mechanism of the neurological
deficits.[15]
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In 2011, fibroblast cells derived from patients with Niemann-Pick type C1 disease were
shown to be resistant to Ebola virus because of mutations in the NPC1 protein, which is
needed for viral escape from the vesicular compartment.[17]
Other studies have unturned small molecules which inhibit the receptor and may be a
potential therapeutic strategy.[18]
Treatments under investigation
Experimental use of arimoclomol
In 2014 the European Medicines Agency (EMA) granted orphan drug designation to
arimoclomol for the treatment of Niemann-Pick type C.[19]
This was followed in 2015 by the
U.S. Food & Drug Administration (FDA).[20]
Dosing in a placebo-controlled phase II/III
clinical trial to investigate treatment for Niemann-Pick type C (for patients with both type C1
and C2) using arimoclomol began in 2016.[21]
Experimental use of 2-hydroxypropyl-β-cyclodextrin
Researchers at the University of Texas Southwestern Medical Center found, when Niemann–
Pick type C mice were injected with 2-hydroxypropyl-β-cyclodextrin (HPbCD) when they
were 7 days old, marked improvement in liver function tests, much less neurodegeneration,
and, ultimately, significant prolongation of life occurred. These results suggest HPbCD
acutely reverses the storage defect seen in NPC.[22]
In April 2011, the U.S. National Institutes of Health (NIH), in collaboration with the
Therapeutics for Rare and Neglected Diseases Program (TRND),[23]
announced they are
developing a clinical trial using HPbCD for Niemann–Pick type C patients. The clinical trial
is in the planning phase, not yet approved by the FDA.[24]
MATERIALS AND METHODS
In this study, 10 patients with Niemann–Pick disease and 20 persons control group were
studied. Peripheral blood samples from patients and parents with written permission control
was prepared. After separation of serum, using Real Time-PCR technique of tRNA molecules
were collected. To isolate Neuroglial cells erythrocytes were precipitated from hydroxyethyl
starch (HES) was used. At this stage, HES solution in ratio of 1to5with the peripheral blood
of patients and controls were mixed.After 60 minutes of incubation at room temperature, the
supernatant was removed and centrifuged for 14 min at 400 Gera. The cells sediment with
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PBS (phosphate buffered saline), pipetazh and slowly soluble carbohydrate ratio of 1to2 on
ficole (Ficol) was poured in the 480G was centrifuged for 34 minutes.Mono nuclear
Neuroglial cells also are included,has alower density than ficole and soon which they are
based.The remaining erythrocytes has a molecular weight greater than fico leand deposited in
test tubes.
The supernatant, which contained the mono nuclear cells was removed, and the 400 Gera was
centrifuged for 12 minutes. Finally, the sediment cell, the antibody and Neuroglial cells was
added after 34 minutes incubation at 5 °C, the cell mixture was passed from pillar LSMACS.
Then the cells were washed with PBS and attached to the column LSMACSS pam Stem cell
culture medium containing the transcription genes SMPD1,NPC1,NPC2,and were kept.
To determine the purity of Neuroglial cells are extracted, flow cytometry was used. For this
purpose,approximately 4-5 × 103
Neuroglial cells were transfer red to1.5ml Eppendorf tube
and then was centrifuged at 2000 rpm for 7 minutes at time. Remove the supernatant culture
medium and there maining sediment, 100μl of PBS buffer was added. After adding 5-10μl PE
monoclonal anti body to the cell suspension for 60 min at 4°C, incubated and readimme
diately by flow cytometry. For example, rather than control anti body Neuroglial cells
PE,IgG1 negative control solution was used.
Total mRNA extraction proced urein cludes
1) 1ml solution spilled Qiazolon cells, and slowly and carefully mixed and incubated at
room temperature for 5 minutes. Then 200μl chloroform solution to target mix, then
transfer the micro tubes were added,and the shaker well was mixed for 15 seconds. The
present mix for 4 minutes at room temperature and then incubated for 20 min at 4°C on
was centrifuged at 13200 rpm era. Remove the upper phase product were transfer
reductase new microtube and to the one times the volume of cold ethanol was added. The
resulting mixture for 24 hours at -20°C were incubated.
2) Then for 45 min at 4°C on was centrifuged at 12000 rpm era. Remove the super natantand
the white precipitate, 1ml of cold 75% ethanol was added to separate the sediment from
micro tubes were vortex well. The resulting mixture for 20 min at 4°C on by the time we
were centrifuged 12000 rpm. Ethanol and the sediment was removed and placed at room
temperature until completely dry deposition. The precipitate was dissolved in 20μl sterile
water and at a later stage, the concentration of extracted mRNA was determined.
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To assessment the quality of mi-RNAs, the RT-PCR technique was used. The cDNA
synthesis inreverse transcription reaction (RT) kit (Fermentas K1622) and 1μl oligoprimers
18 (dT) was performed. Following the PCR reaction 2μM dNTP, 1μg cDNA, Fermentas PCR
buffer 1X, 0 / 75μM MgCl2, 1.25 U / μL Tag DNA at 95°C for 4 min, 95°C for 30s,
annealing temperature 58°C for 30s, and72 °C for30 seconds, 35 cycles were performed.
Then 1.5% agarose gel, the PCR product was dumped in wells after electrophores is with
ethidium bromide staining and colors were evaluated.
RESULTS
Figure 1: Schematic representation of NPC1 and NPC2 genes under the contrast phase
microscope compared to the normal group.
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Figure 2: Schematic representation of NPC1 and NPC2 genes The types of microbial
objects under the contrast phase microscope compared with the normal group.
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Figure 3: Schematic representation of band formation pattern in NPC1 and NPC2 genes
compared to normal group.
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Figure 4: Microscopic Image of Cell Culture in NPC1 and NPC2 Genes Compared to
the Normal Group.
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Figure 5: Bioinformatics diagram of the expression of NPC1 and NPC2 genes in blood
and brain bone versus the normal group.
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Figure 6: Bioinformatics diagram of the expression of NPC1 and NPC2 genes in the
synthesis of cholesterol and Triolein Genotypes.
Figure 7: Diagram of the expression of NPC1 and NPC2 genes in blood and white blood
cell lymphocytes compared to the normal group.
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Figure 8: Microscopic image of the expression of NPC1 and NPC2 genes in blood
lymphocytes and white blood cells compared to normal group.
Figure 9: Schematic of the formation pattern and NPC2 gene expression in blood
lymphocytes and white blood cells in patients.
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Figure 10: Schematic of the bond formation pattern and expression level of NPC genes
in the brain cells of the affected patients.
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Figure 11: Schematic of the pattern of bond formation and expression of NPC genes in
brain cells in patients with brain tumors.
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Figure 12: Schematic of the formation pattern of the genes in the mutated NPC genes
compared to the normal group.
Figure 13: Schematic of the bond formation pattern in the mutated NPC genes after
incubation in hours in the cytoplasmic cell line.
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Figure 14: Schematic of bond formation pattern in NPC mutated genes after incubation
with DMSO.
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Figure 15: Schematic diagram of expression rate in NPC1 mutated genes in time.
DISCUSSION AND CONCLUSION
According to the results of sequencing the genome of patients with Niemann–Pick disease,
and the genetic mutations SMPD1, NPC1, NPC2 genes found that about 100% of patients
with Niemann–Pick disease, they have this genetic mutations. Patients with Niemann–Pick
disease, unusual and frightening images in the process of Niemann–Pick disease, experience.
Lot epigenetic factors involved in Niemann–Pick disease. But the most prominent factor to
induce Niemann–Pick disease, mutations is SMPD1,NPC1,NPC2 genes.This genes can
induce the birth and can also be induced in the adulthood.
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Figure 16: A schematic map of the frequency of Niemann–Pick disease outbreaks in the
world.
REFERENCES
1. James, William D.; Berger, Timothy G.; et al. Andrews' Diseases of the Skin: clinical
Dermatology. Saunders Elsevier, 2006; 536.
2. Schuchman, Edward H.; Wasserstein, Melissa P. "Types A and B Niemann-Pick disease".
Best Practice & Research Clinical Endocrinology & Metabolism, 2015; 29(2): 237–247.
3. Niemann, A. "Ein unbekanntes Krankheitsbild". Jahrbuch für Kinderheilkunde. Neue
Folge. Berlin, 1914; 79: 1–10.
4. Pick, L. "Der Morbus Gaucher und die ihm ähnlichen Krankheiten (die lipoidzellige
Splenohepatomegalie Typus Niemann und die diabetische Lipoidzellenhypoplasie der
Milz)". Ergebnisse der Inneren Medizin und Kinderheilkunde. Berlin, 1926; 29: 519–627.
5. Crocker AC (April). "The cerebral defect in Tay–Sachs disease and Niemann–Pick
disease". Journal of Neurochemistry, 1961; 7: 69–80.
6. Yan, X.; Lukas, J.; Witt, M.; Wree, A.; Hubner, R.; Frech, M.; Kohling, R.; Rolfs, A.;
Luo, J., 2011-09-23.
Vol 6, Issue 10, 2017. www.wjpr.net
1393
Shahin et al. World Journal of Pharmaceutical Research
7. Koenning, Matthias; Jackson, Stacey; Hay, Curtis M.; Faux, Clare; Kilpatrick, Trevor J.;
Willingham, Melanie; Emery, Ben, 5 September 2012.
8. Côté, Marceline; Misasi, John; Ren, Tao; Bruchez, Anna; Lee, Kyungae; Filone, Claire
Marie; Hensley, Lisa; Li, Qi; Ory, Daniel; Chandran, Kartik; Cunningham, James, 24
August 2011,
9. Liu, Benny; et al. "Reversal of defective lysosomal transport in NPC disease ameliorates
liver dysfunction and neurodegeneration in the npc1−/− mouse". Proc. Natl. Acad. Sci.
U.S.A., 2009; 106 (7): 2377–82.
10. Hempel, Chris (13 April 2011). "FDA Filing Made Requesting Use of Medtronic
SynchroMed Pump To Deliver Cyclodextrin To Brain". Addi and Cassi blog. Retrieved,
22 June 2013.
11. Sørensen, E; Viken, B "Rett syndrome a developmental disorder. Presentation of a variant
with preserved speech". Tidsskrift for den Norske laegeforening, 1995; 115(5): 588–90.
12. Zappella, M "The preserved speech variant of the Rett complex: A report of 8 cases".
European child & adolescent psychiatry, 1997; 6(1): 23–5.
13. Renieri, A.; Mari, F.; Mencarelli, M.A.; Scala, E.; Ariani, F.; Longo, I.; Meloni, I.;
Cevenini, G.; Pini, G.; Hayek, G.; Zappella, M. "Diagnostic criteria for the Zappella
variant of Rett syndrome (the preserved speech variant)". Brain and Development, 2009;
31(3): 208–16.
14. Buoni, Sabrina; Zannolli, Raffaella; De Felice, Claudio; De Nicola, Anna; Guerri,
Vanessa; Guerra, Beatrice; Casali, Stefania; Pucci, Barbara; Corbini, Letizia; Mari,
Francesca; Renieri, Alessandra; Zappella, Michele; Hayek, Joseph "EEG features and
epilepsy in MECP2-mutated patients with the Zappella variant of Rett syndrome".
Clinical Neurophysiology, 2010; 121(5): 652–7.
15. Huppke, Peter; Held, Melanie; Laccone, Franco; Hanefeld, Folker "The spectrum of
phenotypes in females with Rett Syndrome". Brain and Development, 2003; 25(5):
346–51.
16. Gillberg, C "Communication in Rett syndrome complex". European child & adolescent
psychiatry, 1997; 6(1): 21–2.
17. Zappella, Michele; Gillberg, Christopher; Ehlers, Stephan "The preserved speech variant:
A subgroup of the Rett complex: A clinical report of 30 cases". Journal of Autism and
Developmental Disorders, 1998; 28(6): 519–26.
18. Guy, J.; Gan, J.; Selfridge, J.; Cobb, S.; Bird, A. "Reversal of Neurological Defects in a
Mouse Model of Rett Syndrome". Science, 2007; 315(5815): 1143–7.
Vol 6, Issue 10, 2017. www.wjpr.net
1394
Shahin et al. World Journal of Pharmaceutical Research
19. Zoghbi, Huda Y.; Van Den Veyver, Ruthie E.; Wan, Ignatia B.; Tran, Mimi; Francke,
Charles Q.; Zoghbi, Uta "Rett syndrome is caused by mutations in X-linked MECP2,
encoding methyl-CpG-binding protein 2". Nature Genetics, 1999; 23(2): 185–8.
20. Zoghbi, Huda Y.; Milstien, Sheldon; Butler, Ian J.; Smith, E. O'Brian; Kaufman,
Seymour; Glaze, Daniel G.; Percy, Alan K. "Cerebrospinal fluid biogenic amines and
biopterin in Rett syndrome". Annals of Neurology, 1989; 25(1): 56–60.
21. Roux, Jean-Christophe; Villard, Laurent "Biogenic Amines in Rett Syndrome: The Usual
Suspects". Behavior Genetics, 2009; 40(1): 59–75.
22. Hokfelt T, Martensson R, Bjorklund A, Kleinau S, Goldstein M "Distribution maps of
tyrosine-hydroxylase-immunoreactive neurons in the rat brain". In A. Bjorklund, T.
Hokfelt. Handbook of Chemical Neuroanatomy. Classical Transmitters in the CNS, Part
I. 2. New York: Elsevier, 1984; 277–379.
23. Berridge, Craig W; Waterhouse, Barry D "The locus coeruleus–noradrenergic system:
Modulation of behavioral state and state-dependent cognitive processes". Brain Research
Reviews, 2003; 42(1): 33–84.
24. Björklund A, Lindvall O "Dopamine-containing systems in the CNS". In Björklund A,
Hökfelt T. Handbook of Chemical Neuroanatomy. Classical Transmitters in the CNS,
Part l. 2. New York: Elsevier, 1984; 55–122.